Glucagon-Like Peptide-1 derivatives and their pharmaceutical use

ABSTRACT

The invention relates to protracted Glucagon-Like Peptide-1 (GLP-1) derivatives and therapeutic uses thereof. The GLP-1 derivative of the invention comprises a modified GLP-1(7-37) sequence having a total of 2-12 amino acid modifications, including Glu22 and Arg26, and being derivatised with an albumin binding residue or pegylated in position 18, 20, 23, 30, 31, 34, 36, 37, or 39. These compounds are useful in the treatment or prevention of diabetes type 2 and related diseases. The compounds are potent, stable, have long half-lives, a high affinity of binding to albumin, and/or a high affinity of binding to the extracellular domain of the GLP-1 receptor (GLP-1R), all of which is of potential relevance for the overall aim of achieving long-acting, stable and active GLP-1 derivatives with a potential for once weekly administration.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 U.S.C. §371 national stage application ofInternational Patent Application PCT/EP2008/061755 (published as WO2009/030738), filed Sep. 5, 2008, which claimed priority of EuropeanPatent Applications 07115746.5, filed Sep. 5, 2007 and 08101008.4, filedJan. 28, 2008; this application further claims priority under 35 U.S.C.§119 of U.S. Provisional Applications 60/971,930, filed Sep. 13, 2007and 61/024,345, filed Jan. 29, 2008.

FIELD OF THE INVENTION

This invention relates to the field of therapeutic peptides, i.e. to newprotracted peptide derivatives of Glucagon-Like Peptide-1 (GLP-1).

INCORPORATION-BY-REFERENCE OF THE SEQUENCE LISTING

In accordance with 37 C.F.R. §1.52(e)(5), Applicants enclose herewiththe Sequence Listing for the above-captioned application entitled“SEQUENCE LISTING”, created on Feb. 4, 2010. The Sequence Listing ismade up of 6 kilobytes, and the information contained in the attached“SEQUENCE LISTING” is identical to the information in the specificationas originally filed. No new matter is added.

BACKGROUND OF THE INVENTION

A range of different approaches have been used for modifying thestructure of GLP-1 compounds in order to provide a longer duration ofaction in vivo.

WO 2006/097535 discloses various peptide agonists of the glucagon familywith secretin like activity and their therapeutic use. GLP-1(7-37)derivatives comprising a modified GLP-1(7-37) sequence are disclosed inExamples 3 and 5 thereof. These derivatives, however, do not have a Gluresidue at position 22 and an Arg residue at position 26.

WO 01/04156 discloses peptides that lower blood glucose levels.Compounds 4, 5, 6, 7, 10, 11, 12, and 13 thereof are GLP-1 derivatives,however none of these compounds have a Glu residue at position 22 and anArg residue at position 26.

EP 1364967 discloses glucagon-like insulinotropic peptides, compositionsand methods. None of the five GLP-1 compositions of Example 1 thereofcomprises a modified GLP-1 sequence having Glu at position 22 and Arg atposition 26.

WO 98/08871 discloses protracted GLP-1 derivatives includingliraglutide, which is a GLP-1 derivative for once daily administrationdeveloped by Novo Nordisk A/S. Liraglutide is expected to be marketedfor the treatment of type 2 diabetes as of 2009. Liraglutide does nothave Glu at position 22 and Arg at position 26. The derivative disclosedin Example 30 of this reference does, but is not derivatised in position18, 20, 23, 30, 31, 34, 36, 37, or 39.

WO 2005/027978 discloses novel GLP-1 derivatives including three whichcomprise a modified GLP-1(7-37) sequence having a Glu at position 22 andan Arg at position 26. These derivatives, however, are not derivatisedin position 18, 20, 23, 30, 31, 34, 36, 37, or 39.

Runge et al (Journal of Biological Chemistry, vol. 283, no. 17, pp.11340-11347), which was published after the priority dates of thepresent invention, discloses the crystal structure of the extracellulardomain of the ligand-bound GLP-1 receptor.

Many diabetes patients particularly in the type 2 diabetes segment aresubject to so-called “needle-phobia”, i.e. a substantial fear ofinjecting themselves. In the type 2 diabetes segment most patients aretreated with oral hypoglycemic agents, and since GLP-1 compounds areexpected to be an injectable pharmaceutical product these patients willbe administered, the fear of injections may become a serious obstaclefor the widespread use of the clinically very promising compounds. Thus,there is a need to develop new compounds which can be administered lessthan once daily, e.g. once every second or third day preferably onceweekly, while retaining an acceptable clinical profile or optionally vianon invasive administration such as pulmonary, nasal, sublingual, bucalor oral administration.

One object of the present invention is to provide a chemically,physically and enzymatically stable GLP-1 derivative, preferably with ahigh alpha-helical content.

A further object of the invention is to provide a long acting, i.e.having an administration regimen as described above, GLP-1 derivative.

Another object of this invention is to provide a GLP-1 derivative withhigh potency (receptor affinity) in order to reduce the therapeutic doseused for example for once weekly s.c. dosing or alternatively fornon-invasive delivery.

Another object of this invention is to provide a GLP-1 derivative with ahigh binding affinity to the extracellular domain of the GLP-1 receptor(GLP-1R).

Another object of this invention is to provide a GLP-1 derivative withhigh albumin binding affinity which protects the peptide for proteolyticdegradation and reduce renal clearance of the peptide.

Potency, stability, half-life, binding affinity to albumin, and bindingaffinity to the extracellular domain of the GLP-1 receptor areproperties of potential relevance for an overall object of achievinglong-acting, stable and of course therapeutically active GLP-1derivatives with a potential for once weekly administration.

SUMMARY OF THE INVENTION

In one aspect of the invention, a GLP-1 derivative is provided whichcomprises a modified GLP-1(7-37) sequence having: i) a total of 2-12amino acid modifications as compared to GLP-1(7-37) (SEQ ID No: 1),including a) a Glu residue at a position equivalent to position 22 ofGLP-1(7-37), and b) an Arg residue at a position equivalent to position26 of GLP-1(7-37); and ii) which is derivatised with an albumin bindingresidue or pegylated in a position selected from a position equivalentto position 18, 20, 23, 30, 31, 34, 36, 37, or 39 of GLP-1(7-37).

In a further aspect, pharmaceutical compositions and methods and uses ofthe derivatives according to the invention, are provided.

DESCRIPTION OF THE INVENTION Definitions and Preferred Embodiments

In the present specification, the following terms have the indicatedmeaning:

The term “polypeptide” and “peptide” as used herein means a compoundcomposed of at least five constituent amino acids connected by peptidebonds. The constituent amino acids may be from the group of the aminoacids encoded by the genetic code and they may be natural amino acidswhich are not encoded by the genetic code, as well as synthetic aminoacids. Natural amino acids which are not encoded by the genetic code aree.g., γ-carboxyglutamate, ornithine, phosphoserine, D-alanine andD-glutamine. Synthetic amino acids comprise amino acids manufactured bychemical synthesis, i.e. D-isomers of the amino acids encoded by thegenetic code such as D-alanine and D-leucine, Aib (α-aminoisobutyricacid), Abu (α-aminobutyric acid), Tle (tert-butylglycine), β-alanine,3-aminomethyl benzoic acid, anthranilic acid.

The 22 proteogenic amino acids are: Alanine, Arginine, Asparagine,Aspartic acid, Cysteine, Cystine, Glutamine, Glutamic acid, Glycine,Histidine, Hydroxyproline, Isoleucine, Leucine, Lysine, Methionine,Phenylalanine, Proline, Serine, Threonine, Tryptophan, Tyrosine, Valine.

Thus a non-proteogenic amino acid (which may also be designated anon-natural amino acid) is a moiety which can be incorporated into apeptide via peptide bonds but is not a proteogenic amino acid. Examplesare γ-carboxyglutamate, ornithine, phosphoserine, the D-amino acids suchas D-alanine and D-glutamine, Synthetic non-proteogenic amino acidscomprising amino acids manufactured by chemical synthesis, i.e.D-isomers of the amino acids encoded by the genetic code such asD-alanine and D-leucine, Aib α-aminoisobutyric acid), Abu(α-aminobutyric acid), Tle (tert-butylglycine), 3-aminomethyl benzoicacid, anthranilic acid, des-amino-histidine (abbreviated DesaminoHis,alternative name imidazopropionic acid, abbreviated Impr), the betaanalogs of amino acids such as β-alanine etc., D-histidine,2-amino-histidine, β-hydroxy-histidine, homohistidine,N^(α)-acetyl-histidine, β-fluoromethyl-histidine, α-methyl-histidine,α,α-dimethyl-glutamic acid, m-CF₃-phenylalanine (abbreviated m-CF₃-Phe),α,β-diaminopropionic acid (abbreviated Dap), 3-pyridylalanine,2-pyridylalanine or 4-pyridylalanine, (1-aminocyclopropyl) carboxylicacid, (1-aminocyclobutyl) carboxylic acid, (1-aminocyclopentyl)carboxylic acid, (1-aminocyclohexyl) carboxylic acid,(1-aminocycloheptyl) carboxylic acid, or (1-aminocyclooctyl) carboxylicacid.

The term “analogue” as used herein referring to a polypeptide means amodified peptide wherein one or more amino acid residues of the peptidehave been substituted by other amino acid residues and/or wherein oneacid amino residue has been deleted from the peptide in the C-terminalof the peptide or wherein one amino acid residue has been added to theC-terminal of the peptide.

The term “modified peptide” as used herein refers to a modified peptideas defined above. For the present purposes this term is usedinterchangeably with the term “modified peptide sequence”. Consistentlyherewith, the term “modification” when used herein in connection withpeptide sequences refers to amino acid substitutions, additions, and/ordeletions.

For the present purposes any amino acid substitution, deletion, and/oraddition refers to the sequence of human GLP-1(7-37) which is includedherein as SEQ ID No: 1. However, the numbering of the amino acidresidues in the sequence listing always starts with no. 1, whereas forthe present purpose we want, following the established practice in theart, to start with amino acid residue no. 7 and assign number 7 to it.Therefore, generally, any reference herein to a position number of theGLP-1(7-37) sequence is to the sequence starting with His at position 7and ending with Gly at position 37.

A simple system is often used to describe analogues: For example[Arg³⁴]GLP-1(7-37)Lys designates a GLP-1(7-37) analogue wherein thenaturally occurring lysine at position 34 has been substituted witharginine and wherein a lysine has been added to the C-terminal aminoacid residue, i.e. to the Gly³⁷. This GLP-1 analogue accordingly has twoamino acid modifications as compared to GLP-1(7-37), viz. onesubstitution and one addition.

The expression “a position equivalent to” when used herein tocharacterize a modified GLP-1(7-37) sequence refers to the correspondingposition in the natural GLP-1(7-37) sequence (having the sequence of SEQID No: 1). Corresponding positions are easily deduced, e.g. by simplehandwriting and eyeballing. In the alternative, a standard protein orpeptide alignment program may be used, such as “align” which is aNeedleman-Wunsch alignment. The algorithm is described in Needleman, S.B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48: 443-453,and the align program by Myers and W. Miller in “Optimal Alignments inLinear Space” CABIOS (computer applications in the biosciences) (1988)4:11-17. For the alignment, the default scoring matrix BLOSUM50 and thedefault identity matrix may be used, and the penalty for the firstresidue in a gap may be set at −12 and the penalties for additionalresidues in a gap at −2.

As another example, the GLP-1 derivative of Example 1 herein comprisesthe following modified GLP-1(7-37) sequence:[desaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1(7-37)amide, which has atotal of 5 amino acid modifications (in this case all substitutions),including a Glu at a position equivalent to position 22 of GLP-1(7-37),and an Arg at a position equivalent to position 26 of GLP-1(7-37).

All amino acids for which the optical isomer is not stated is to beunderstood to mean the L-isomer.

The term “derivative” as used herein in relation to a peptide means achemically modified peptide or an analogue thereof, wherein at least onesubstituent is not present in the unmodified peptide or an analoguethereof, i.e. a peptide which has been covalently modified. Typicalmodifications are amides, carbohydrates, alkyl groups, acyl groups,esters and the like.

The GLP-1 derivatives of the invention are derivatised with an albuminbinding residue or pegylated in a position selected from position 18,20, 23, 30, 31, 34, 36, 37, or 39. The derivatisation refers to acovalent link as explained above.

For example a lysine residue or cysteine residue may be linked to analbumin binding residue via a chemical bond. Such a chemical bond can asan example be obtained by derivatisation of an epsilon amino group oflysine by acylation with an active ester of an albumin binding residuesuch as a long fatty acid.

An example of a GLP-1 derivative of the invention (a derivative of ananalogue of GLP-1(7-37)) isN-epsilon37{2-[2-(2-{2-[2-((R)-3-carboxy-3-{[1-(19-carboxynonadecanoyl)piperidine-4-carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl[desaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1(7-37)amide (the compound ofExample 1 herein). In this compound, the naturally occurring Gly inposition 37 has been substituted with lysine which has been derivatisedat N-epsilon37 with the following albumin binding residue:

-   {2-[2-(2-{2-[2-((R)-3-carboxy-3-{[1-(19-carboxynonadecanoyl)piperidine-4-carbon    yl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl    (structure 1)

and wherein the naturally occurring histidine in position 7 has beensubstituted with desaminoHis and the naturally occurring glycine in pos22 has been substituted with glutamate and lysine in position 26 and 34has been substituted with arginine. In this derivative, the GLP-1(7-37)analogue of [desaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1(7-37)amide hasbeen derivatised (covalently modified with an amide bond) with analbumin binding residue in a position equivalent to position 37 ofGLP-1(7-37), viz. at the epsilon amino group of Lys at position 37.Accordingly, a GLP-1 derivative is a compound with two constituents: AGLP-1 peptide part and a derivative part which are covalently linked toeach other.

The amino acid residue which is derivatised may comprise an amino group.Examples of amino acid residues comprising an amino group is lysine,ornithine, Epsilon-N-alkylated lysine such as Epsilon-N methylLysine,O-aminoethylserine, O-aminopropylserine or longer O alkylated serinescontaining a primary or secondary amino group in the side chain. In afurther aspect of the invention, the derivatised amino acid residuecomprises a primary amino group in a side chain. Examples of amino acidresidues comprising a primary amino group is lysine ornithine,O-aminoethylserine, O-aminopropylserine or longer O alkylated serinescontaining a primary amino group in the side chain. In yet a furtheraspect of the invention, the derivatised amino acid residue is lysine.In yet a further aspect of the invention, the derivative according tothe invention is only derivatised in one position, e.g. only one aminoacid residue is derivatised.

Other examples of connecting two chemical moieties as used in thepresent invention includes but is not limited to alkylation, esterformation, amide formation or maleimide coupling.

The term “GLP-1 peptide” as used herein means GLP-1(7-37) (SEQ ID No: 1)or a GLP-1(7-37) analogue thereof.

The term “GLP-1(7-37)” is intended to include GLP-1(7-37) (i.e. thepeptide), as well as the corresponding amide(s), and the same applies toGLP-1(7-37) analogues.

In a particular embodiment, the GLP-1 derivative of the invention is anamide. In another particular embodiment it is a peptide, i.e. the GLP-1peptide moiety of the derivative has a free carboxylic group at theC-terminus.

The terms “GLP-1(7-34)”, “GLP-1(7-35)”, “GLP-1(7-36)”, “GLP-1(7-38)”,“GLP-1(7-39)”, “GLP-1(7-40)”, “GLP-1(7-41)” or derivative thereof is nowand then used herein to specify the exact length of the GLP-1 peptidemoiety of a derivative of the invention. For example, “a GLP-1(7-34)derivative” refers to a GLP-1(7-37) derivative in which the last threeC-terminal amino acid residues have been deleted. As another example, “aGLP-1(7-39) derivative” refers to a GLP-1(7-37) derivative in which twoamino acid residues have been added to the C-terminus.

In a preferred embodiment, the GLP-1 derivative of the invention has thesequence of formula (I):

Formula (I) (SEQ ID No: 2)Xaa₇-Xaa₈-Xaa₉-Gly-Thr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Tyr-Xaa₂₀-Glu-Glu-Xaa₂₃-Xaa₂₄-Xaa₂₅-Arg-Xaa₂₇-Xaa₂₈-Ile-Xaa₃₀-Xaa₃₁-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-Xaa₄₀-Xaa₄₁-Rwherein(Xaa₇-Xaa₈) is (L-histidine-Aib), (desamino-histidine-alanine), or(desamino-histidine-Aib);Xaa₉ is Glu, or a Glu derivative;Xaa₁₆ is Val, or Leu;Xaa₁₈ is Ser, Lys, Cys, or Arg;Xaa₂₀ is Leu, or Lys;Xaa₂₃ is Gln, Glu, Lys, Cys, or Arg;Xaa₂₄ is Ala, or Asn;Xaa₂₅ is Ala, or Val;Xaa₂₇ is Glu, Ala, or Leu;Xaa₂₈ is Phe, or a Phe derivative;Xaa₃₀ is Ala, Glu, Lys, or Arg;Xaa₃₁ is Trp, Cys, or Lys;Xaa₃₃ is Val, Cys, or Lys;Xaa₃₄ is Lys, Cys, Glu, Asn, Dap, or Arg;Xaa₃₅ is Gly, Arg, Lys, Aib, or absent;Xaa₃₆ is Arg, Lys, or absent;Xaa₃₇ is Gly, Aib, Cys, Lys, epsilon-amino-Lys, Pro, Arg, or absent;Xaa₃₈ is Lys, Glu, Arg, or absent;Xaa₃₉ is Lys, Arg, or absent;Xaa₄₀ is Arg, or absent;Xaa₄₁ is Arg, or absent; andR is amide, or absent;provided that if Xaa₃₇, Xaa₃₈, Xaa₃₉, or Xaa₄₀ is absent, then eachamino acid residue downstream is also absent;and which is derivatised with an albumin binding residue or pegylated ina position selected from a position equivalent to position 18, 20, 23,30, 31, 34, 36, 37, or 39 of GLP-1(7-37) (SEQ ID No: 1).

A non-limiting example of a “Glu derivative” is alpha,alphadimethyl-Glu.

A non-limiting example of “a Phe derivative” is CF₃-Phe, such asm-CF₃-Phe (see e.g. the compound of Example 3).

The term epsilon-amino-Lys (or epsilon-Lys) is intended to indicate thatamino acid residue no. 38, lysine, is bound to the GLP-1(7-37) peptidevia its epsilon amino group, not (as is usually the case) via its alphaamino group (see e.g. the compound of Example 28).

In another preferred embodiment, the GLP-1 derivative of the inventionhas the sequence of formula (II):

Formula (II) (SEQ ID No: 3)Xaa₇-Xaa₈-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Xaa₁₈-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Arg-Glu-Phe-Ile-Xaa₃₀-Xaa₃₁-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇- Xaa₃₈-Xaa₃₉-Xaa₄₀-Xaa₄₁-RwhereinXaa₇ is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine,8-hydroxy-histidine, homohistidine, N^(α)-acetyl-histidine,α-fluoromethyl-histidine, α-methyl-histidine, 3-pyridylalanine,2-pyridylalanine, or 4-pyridylalanine;Xaa₈ is Ala, Gly, Val, Leu, Ile, Lys, Aib, (1-aminocyclopropyl)carboxylic acid, (1-aminocyclobutyl) carboxylic acid,(1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl) carboxylicacid, (1-aminocycloheptyl) carboxylic acid, or (1-aminocyclooctyl)carboxylic acid;Xaa₁₈ is Ser, Lys, or Arg;Xaa₃₀ is Ala, Glu, Lys, or Arg;Xaa₃₁ is Lys, or Trp;Xaa₃₃ is Val, or Lys;Xaa₃₄ is Lys, Glu, Dap, or Arg;Xaa₃₅ is Gly, Arg, Lys, Aib, or absent;Xaa₃₆ is Arg, Lys, or absent;Xaa₃₇ is Gly, Aib, Lys, epsilon-amino-Lys, Pro, Arg, or absent;Xaa₃₈ is Lys, Glu, Arg, or absent;Xaa₃₉ is Lys, Arg, or absent;Xaa₄₀ is Arg, or absent;Xaa₄₁ is Arg, or absent; andR is amide, or is absent;provided that if Xaa₃₇, Xaa₃₈, Xaa₃₉, or Xaa₄₀ is absent, then eachamino acid residue downstream is also absent;and which is derivatised with an albumin binding residue or pegylated ina position selected from a position equivalent to position 18, 20, 23,30, 31, 34, 36, 37, or 39 of GLP-1(7-37) (SEQ ID No: 1).

The term “linker” as used herein means a spacer (the two terms spacerand linker is used interchangeably in the present specification) thatseparates a peptide and an albumin binding residue or a polyethyleneglycol polymer.

The term “pharmaceutically acceptable” as used herein means suited fornormal pharmaceutical applications, i.e. giving rise to no seriousadverse events in patients etc.

The term “excipient” as used herein means the chemical compounds whichare normally added to pharmaceutical compositions, e.g. buffers,tonicity agents, preservatives and the like.

The term “effective amount” as used herein means a dosage which issufficient to be effective for the treatment of the patient comparedwith no treatment.

The term “pharmaceutical composition” as used herein means a productcomprising an active derivative according to the invention, or apharmaceutically acceptable salt, amide, alkyl, ester, or the likethereof, together with pharmaceutical excipients such as buffer,preservative, and optionally a tonicity modifier and/or a stabilizer.Thus a pharmaceutical composition is also known in the art as apharmaceutical formulation.

The term “treatment of a disease” as used herein means the managementand care of a patient having developed the disease, condition ordisorder. The purpose of treatment is to combat the disease, conditionor disorder. Treatment includes the administration of the activederivative according to the invention to eliminate or control thedisease, condition or disorder as well as to alleviate the symptoms orcomplications associated with the disease, condition or disorder.

In one aspect of the invention, the linker comprises one or morealkylene glycol units, such as 1 to 5 alkylene glycol units. Thealkylene glycol units are in a further aspect ethylene glycol, propyleneglycol or butylene glycol but can also be higher alkylene glycols.

In another aspect of the invention, the linker is a hydrophilic linkerselected from—(CH₂)_(l)D[(CH₂)_(n)E]_(m)(CH₂)_(p)-Q_(q)-,whereinl, m and n independently are 1-20 and p is 0-10,Q is —Z—(CH₂)_(l)D[(CH₂)_(n)G]_(m)(CH₂)_(p)—,q is an integer in the range from 0 to 5,each D, E, and G are independently selected from —O—, —NR³—, —N(COR⁴)—,—PR⁵(O)—, and —P(OR⁶)(O)—, wherein R³, R⁴, R⁵, and R⁶ independentlyrepresent hydrogen or C₁₋₆-alkyl,Z is selected from —C(O)NH—, —C(O)NHCH₂—, —OC(O)NH—, —C(O)NHCH₂CH₂—,—C(O)CH₂—, —C(O)CH═CH—, —(CH₂)_(s)—, —C(O)—, —C(O)O— or —NHC(O)—,wherein s is 0 or 1.

In another aspect of the invention, the linker is a hydrophilic linkeras defined above wherein l is 1 or 2, n and m are independently 1-10 andp is 0-10.

In another aspect of the invention, the linker is a hydrophilic linkeras defined above wherein D is —O—.

In another aspect of the invention, the linker is a hydrophilic linkeras defined above wherein E is —O—.

In yet another aspect of the invention, the hydrophilic linker is—CH₂O[(CH₂)₂O]_(m)(CH₂)_(p)Q_(q)-,wherein m is 1-10, p is 1-3, and Q is —Z—CH₂O[(CH₂)₂O]_(m)(CH₂)_(p)—wherein Z is as defined above.

In another aspect of the invention, the linker is a hydrophilic linkeras defined above wherein q is 1.

In another aspect of the invention, the linker is a hydrophilic linkeras defined above wherein G is —O—.

In another aspect of the invention, the linker is a hydrophilic linkeras defined above wherein Z is selected from the group consisting of—C(O)NH—, —C(O)NHCH₂—, and —OC(O)NH—.

In another aspect of the invention, the linker is a hydrophilic linkeras defined above wherein q is 0.

In another aspect of the invention, the linker is a hydrophilic linkeras defined above wherein l is 2.

In another aspect of the invention, the linker is a hydrophilic linkeras defined above wherein n is 2.

In one aspect of this invention a “hydrophilic linker” is used thatseparates a peptide and an albumin binding residue with a chemicalmoiety.

In one aspect of this invention, the hydrophilic linker is—C(O)—(CH₂)_(l)—O—[(CH₂CH₂—O]_(m)—(CH₂)_(p)—NHC(O)—(CH₂)_(l)—O—[(CH₂)_(n)—O]_(m)—(CH₂)_(q)—NH—,wherein l, m, n, and p independently are 1-5, and q is 0-5.

In yet another aspect of this invention, the hydrophilic linker is—C(O)—CH₂—O—CH₂CH₂—O—CH₂CH₂[NHC(O)—CH₂—O—CH₂CH₂O—CH₂CH₂]_(q)—NH—,wherein q is 0-5.

In yet another aspect of this invention, the hydrophilic linker is—C(O)—CH₂—O—CH₂CH₂—O—CH₂CH₂—NHC(O)—CH₂—O—CH₂CH₂O—CH₂CH₂—NH—.

In yet another aspect of the invention, the hydrophilic linker is—[CH₂CH₂O]_(m+1)(CH₂)_(p)Q_(q)- wherein m and p independently is 0-10,and

Q is —Z—(CH₂)_(l)D[(CH₂)_(n)G]_(m)(CH₂)_(p)— as defined above.

In yet another aspect of the invention, the hydrophilic linker is—(CH₂)_(l)—O—[(CH₂)_(n)—O]_(m)—(CH₂)_(p)—[C(O)NH—(CH₂)_(l)—O—[(CH₂)_(n)—O]_(m)—(CH₂)_(p)]_(q)—,wherein l, m, n, and p independently are 1-5, and q is 0-5.

In a further aspect of the invention, the linker comprises an amino acidresidue except Cys, or a dipeptide such as Gly-Lys. In the present text,the expression “a dipeptide such as Gly-Lys” is used to designate adipeptide wherein the C-terminal amino acid residue is Lys, His or Trp,preferably Lys, and wherein the N-terminal amino acid residue isselected from the group comprising Ala, Arg, Asp, Asn, Gly, Glu, Gln,Ile, Leu, Val, Phe and Pro.] Suitable PEG polymers are typicallycommercially available or may be made by techniques well-known to thoseskilled in the art.

In one aspect of the invention, the GLP-1 derivative has been pegylated.

In a particular embodiment, the PEG polymer has a molecular weight ofgreater than 700 D, in a further embodiments a molecular weight greaterthan 5 kD, greater than 10 kD, and greater that 20 kD. The PEG polymermay be linear or branched. In cases where the PEG polymer is greaterthan 20 KDa, the PEG polymer is preferable having a branched structure,such as for example, a 43 kD branched PEG-peptide (Shearwater 2001catalog #2D3XOT01, mPEG2-MAL).

The attachment of a PEG on an intact peptide can be accomplished byattaching the PEG on the opposite side of the peptide surface thatinteracts with the receptor.

There are several strategies for coupling PEG to peptides (see, e.g.Veronese, Biomaterials 22:405-417, 2001), all of which are incorporatedherein by reference in their entirety.

Those skilled in the art, will therefore be able to utilize well-knowntechniques for linking the PEG polymer to GLP-1 peptides describedherein.

Briefly, cysteine PEGylation is one method for site-specific PEGylation,and can be accomplished by introducing a unique cysteine mutation at oneof the specific positions on human amylin or the amylin analog and thenreacting the resulting peptide with a cysteine-specific PEGylationreagent, such as PEG-maleimide. It may be necessary to mutate thepeptide in order to allow for site-specific PEGylation. For example, ifthe peptide contains cysteine residues, these will need to besubstituted with conservative amino acids in order to ensuresite-specific PEGylation. In addition, rigid linkers, including but notlimited to “GGS”, “GGSGGS”, and “PPPS” may be added to the C-terminus,but before the site of PEG attachment (i.e. a unique cysteine residue).

In another aspect, the GLP-1 derivative according to the invention hasbeen derivatised with an albumin binding residue.

In one embodiment, the albumin binding residue is a lipophilic residue.In a further embodiment, the lipophilic residue is attached to a lysineresidue optionally via a linker by conjugation chemistry such as byalkylation, acylation, ester formation, or amide formation or to acysteine residue by maleimide coupling.

In a further embodiment of the invention, the albumin binding residue isnegatively charged at physiological pH. In another aspect of theinvention, the albumin binding residue comprises a group which can benegatively charged. One preferred group which can be negatively chargedis a carboxylic acid group.

In yet another embodiment of the invention, the albumin binding residueis selected from the group consisting of a straight chain alkyl group, abranched alkyl group, a group which has an ω-carboxylic acid group, anda partially or completely hydrogenated cyclopentanophenanthreneskeleton.

In a further embodiment of the invention, the albumin binding residue isa cibacronyl residue.

In a further embodiment of the invention, the albumin binding residuehas from 6 to 40 carbon atoms, from 8 to 26 carbon atoms or from 8 to 20carbon atoms.

In a further embodiment of the invention, the albumin binding residue isan acyl group selected from the group comprising CH₃(CH₂)_(r)CO—,wherein r is an integer from 4 to 38, preferably an integer from 4 to24, more preferred selected from the group comprising CH₃(CH₂)₆CO—,CH₃(CH₂)₈CO—, CH₃(CH₂)₁₀CO—, CH₃(CH₂)₁₂CO—, CH₃(CH₂)₁₄CO—,CH₃(CH₂)₁₆CO—, CH₃(CH₂)₁₈CO—, CH₃(CH₂)₂₀CO— and CH₃(CH₂)₂₂CO—.

In another embodiment of the invention, the albumin binding residue isan acyl group of a straight-chain or branched alkane α,ω-dicarboxylicacid.

In another particular embodiment, the GLP-1 derivative according to theinvention comprises a hydrophilic spacer between the modified GLP-1sequence and one or more albumin binding residue(s).

The hydrophilic spacer may bean unbranched oligo ethylene glycol moietywith appropriate functional groups at both terminals that forms a bridgebetween an amino group of the modified GLP-1 sequence and a functionalgroup of the albumin binding residue.

Preferred embodiments of the GLP-1 derivatives of the invention,compositions including them, and methods of using them are listed in thesections headed “Embodiments according to the invention”, “Additionalembodiments according to the invention”, and “Further particularembodiments according to the invention” close to the start of theexperimental part of the present application.

Functional Properties

A number of GLP-1 derivatives of the invention have been synthesized andtested as described in the experimental part.

The GLP-1 derivatives of the invention have several advantageous andbeneficial properties as explained in the following, by reference to theExamples.

In a first aspect, the GLP-1 derivative of the invention has aprotracted profile of action, which makes it potentially suitable forless-frequently-than-once-daily administration, preferably with apotential for once-weekly, or even less frequent, administration. Theprofile of action can be evaluated in pharmacokinetic experiments withlaboratory animals such as mice or pigs. Suitable experiments are foundin Example 39 (minipigs) and Example 43 (mice) of the presentapplication.

As described in minipig Example 39, (i) the GLP-1 derivative may beadministered s.c. or i.v., preferably s.c.; (ii) the pigs are preferablyGöttingen minipigs, preferably about 5 months old and weighing 8-10 kg;(iii) the animals are preferably fasting before administration,preferably as described; (iv) the injection is preferably given asindicated; (v) the number of animals tested is preferably as indicated;(vi) the dosage is preferably as indicated; and/or (vii) blood samplesare also preferably taken, collected, and assayed as indicated in thisExample.

Pharmacokinetic experiments like the one in Example 39 result in aplasma concentration profile of the compound in question versus time, onthe basis of which the half-life, T½, may be determined, preferably asdescribed in Example 39.

In a first particular embodiment, the half-life of the GLP-1 derivativeof the invention, after s.c. administration in minipigs, is above 18hours, preferably above 24 hours, more preferably above 28 hours, evenmore preferably above 30 hours, most preferably above 32 hours.

In a second particular embodiment, the half-life of the GLP-1 derivativeof the invention, after s.c. administration in minipigs, is above 32hours, preferably above 34 hours, more preferably above 36 hours, evenmore preferably above 38 hours, most preferably above 40 hours.

In a third particular embodiment, the half-life of the GLP-1 derivativeof the invention, after s.c. administration in minipigs, is above 45hours, preferably above 50 hours, more preferably above 55 hours, evenmore preferably above 60 hours, most preferably above 65 hours.

In a fourth particular embodiment, the half-life of the GLP-1 derivativeof the invention, after s.c. administration in minipigs, is above 45hours, preferably above 50 hours, more preferably above 55 hours, evenmore preferably above 60 hours, most preferably above 65 hours.

In a fifth particular embodiment, the half-life of the GLP-1 derivativeof the invention, after s.c. administration in minipigs, is above 70hours, preferably above 75 hours, more preferably above 80 hours, evenmore preferably above 85 hours, most preferably above 90 hours.

In a sixth particular embodiment, the half-life of the GLP-1 derivativeof the invention, after s.c. administration in minipigs, is above 92hours, preferably above 94 hours, more preferably above 96 hours, evenmore preferably above 98 hours, most preferably above 100 hours.

In a seventh particular embodiment, the GLP-1 derivative of theinvention has an in vivo half-life of at least 50 hrs after s.c.administration to mini pigs, and preferably an in vivo half-life of atleast 80 hrs after s.c. administration to mini pigs.

Accordingly, exemplary half-life intervals (time indicated in hours, h)of the GLP-1 derivative of the invention, determined in minipigs by s.c.administration, are: 20-100, 30-100, 40-100, 50-100, 60-100, 70-100,80-100, or 90-100 (hours).

The half-life of the GLP-1 derivative of the invention may also bedetermined in a dose-response study in db/db mice, e.g. as described inExample 43. As described in this Example, (i) the mice are preferablyfrom Taconic; (ii) of 10-12 weeks of age; (iii) have free access tostandard feed such as Altromin 1324 and tap water; (iv) are kept at 24dgC; (v) being acclimatised for 1 week; (vi) allocated into 7 groups(preferably n=6) based on matching mean blood glucose values; (vii)receive treatment as described in the example; (viii) being dosed asdescribed; (ix) blood glucose is assessed according to a scheme asdescribed, preferably assayed as described; and/or (x) the half-lifedetermined based on the blood glucose vs. time determinations,preferably as described in the example.

In a first particular embodiment, the half-life of the GLP-1 derivativeof the invention, after s.c. administration in db/db mice, is above 10hours, preferably above 11 hours, more preferably above 12 hours, evenmore preferably above 13 hours, most preferably above 14 hours.

In a second particular embodiment, the half-life of the GLP-1 derivativeof the invention, after s.c. administration in db/db mice, is above 15hours, preferably above 16 hours, more preferably above 17 hours, evenmore preferably above 18 hours, most preferably above 19 hours.

In a third particular embodiment, the half-life of the GLP-1 derivativeof the invention, after s.c. administration in db/db mice, is above 20hours, preferably above 21 hours, more preferably above 22 hours, evenmore preferably above 23 hours, most preferably above 24 hours.

In a fourth particular embodiment, the half-life of the GLP-1 derivativeof the invention, after s.c. administration in db/db mice, is above 25hours, preferably above 26 hours, more preferably above 27 hours, evenmore preferably above 28 hours, most preferably above 29 hours.

Accordingly, exemplary half-life intervals (time indicated in hours, h)of the GLP-1 derivative of the invention, assayed in db/db mice by s.c.administration, are: 5-30, 10-30, 15-30, 20-30, or 25-30 (hours).

In a fifth particular embodiment, the present invention relates to aderivative of a GLP-1 peptide which has substantially improved terminalhalf-life in rodent and in a non-rodent model relative to liraglutide.The terminal half-life in rodent or in a non-rodent model is preferablyimproved at least 3 fold relative to liraglutide. Alternatively, theterminal half-life in a non-rodent model is improved at least 6 foldrelative to liraglutide, or the GLP-1 derivative of the invention has anin vivo half-life of at least 10 hrs after i.v. administration to rats.

In a second aspect, the GLP-1 derivative of the invention has animproved stability. In particular, it has a secondary structure with asignificant alpha-helical stretch. Secondary structures with significantalpha-helical stretch are expected to confer chemical, physical and/orenzymatical stability to the molecule in question.

The alpha-helix content may be determined using circular dichroism (CD)spectroscopy, e.g. as described in Example 44. In particularembodiments, (i) far-UV CD spectra are recorded on 5 uM solutions of thecompound in question, preferably in 10 mM Tris/ClO₄ pH 8.0; (ii) bufferbackground is subtracted; (iii) the data are normalised to molarellipticity M⁻¹ cm⁻¹ based on the concentration of peptide bonds; (iv)the intensity value (delta epsilon) at 222 nm is extracted; (v) thealpha-helical content is calculated based on the intensity value of(iv), assuming proportionality and using for conversion the fact that anintensity value of −1 M⁻¹ cm⁻¹ corresponds to 10% alpha-helicalstructure.

In a first particular embodiment, the GLP-1 derivative of the inventionhas an alpha-helix content of above 20%, preferably above 25%, morepreferably above 30%, even more preferably above 35%, and mostpreferably above 36%.

In a second particular embodiment, the GLP-1 derivative of the inventionhas an alpha-helix content of above 40%, preferably above 45%, morepreferably above 50%, even more preferably above 55%, and mostpreferably above 58%.

Accordingly, exemplary ranges of alpha-helix content of GLP-1derivatives of the invention are 20-60, 30-60, 40-60, and 50-60(%).

In a third particular embodiment the GLP-1 derivative of the inventionis stable against the chemical degradation normally seen withexendin-4—especially oxidation and deamidation.

In a fourth particular embodiment, the GLP-1 derivative of the inventionis chemically and physically stable at neutral pH, most preferably inthe range 6-8.

In a fifth particular embodiment, the GLP-1 derivative of the inventionhas little or no tendency to aggregate. The aggregation tendency ispreferably significantly improved relatively to the aggregation tendencyof liraglutide when tested in a thioflavin assay.

In a third aspect, the GLP-1 derivative of the invention has anacceptable, preferably a high potency (at the receptor). The potency ofan insulinotropic agent such as the GLP-1 derivative of the inventionmay be determined by calculating the EC₅₀ value from the dose-responsecurve as described in Example 40.

The term “insulinotropic agent” as used herein means a derivative whichis an agonist of the human GLP-1 receptor, i.e. a derivative whichstimulates the formation of cAMP in a suitable medium containing thehuman GLP-1 receptor (one such medium disclosed below).

In particular embodiments, (i) baby hamster kidney (BHK) cellsexpressing the cloned human GLP-1 receptor are used, preferablyBHK-467-12A, more preferably BHK-467-12A (tk-ts13); (ii) the cells aregrown in DMEM media with the addition of 100 IU/mL penicillin, 100 μg/mLstreptomycin (1% Pen/Strep), 5% fetal calf serum (FCS) and 0.5 mg/mLGeneticin G-418 (Life Technologies), preferably at 5% CO₂; (iii) thecells, preferably at approximately 80% confluence, are washed twice inphosphate buffered saline (PBS); (iv) the cells are harvested with anaqueous solution of the tetrasodium salt of ethylenediaminetetraaceticacid, such as Versene; (v) plasma membranes are prepared from the cellsby homogenisation, preferably in buffer 1; (vi) the homogenate iscentrifuged, e.g. at 48,000×g for 15 min at 4° C.; and/or (vii) thepellet is suspended by homogenization in buffer 2; Steps (vi) and (viii)are preferably repeated, e.g. one or two times more.

The functional receptor assay may be carried out as described in Example40 by measuring cyclic AMP (cAMP) as a response to stimulation by theinsulinotropic agent. cAMP formed is preferably quantified by theAlphaScreen™ cAMP Kit (Perkin Elmer Life Sciences). Incubations may becarried out in half-area 96-well microtiter plates in a total volume of50 μL buffer 3 (50 mM Tris-HCI, 5 mM HEPES, 10 mM MgCl₂, pH 7.4) andwith the following additions: 1 mM ATP, 1 μM GTP, 0.5 mM3-isobutyl-1-methylxanthine (IBMX), 0.01% Tween-20, 0.1% BSA, 6 μgmembrane preparation, 15 μg/mL acceptor beads, 20 μg/mL donor beadspreincubated with 6 nM biotinyl-cAMP. Derivatives to be tested foragonist activity are preferably dissolved and diluted in buffer 3. GTPis freshly prepared for each experiment. The plate is incubated in thedark with slow agitation for three hours at room temperature followed bycounting in the Fusion™ instrument (Perkin Elmer Life Sciences).Concentration-response curves are plotted for the individual derivativesand EC₅₀ values estimated using a four-parameter logistic model withPrism, preferably in version 4.0, or 5.0, (GraphPad, Carlsbad, Calif.).

In a first particular embodiment, the GLP-1 derivative of the inventionhas a potency (EC₅₀ in nM), as determined using the cAMP assay, below4.00, preferably below 3.50, more preferably below 3.00, even morepreferably below 2.50, and most preferably below 2.00 (nM).

In a second particular embodiment, the GLP-1 derivative of the inventionhas a potency (EC₅₀ in nM), as determined using the cAMP assay, below1.80, preferably below 1.60, more preferably below 1.40, even morepreferably below 1.20, and most preferably below 1.00 (nM).

In a third particular embodiment, the GLP-1 derivative of the inventionhas a potency (EC₅₀ in nM), as determined using the cAMP assay, below0.80, preferably below 0.60, more preferably below 0.40, even morepreferably below 0.20, and most preferably below 0.10 (nM).

In a fourth particular embodiment, the GLP-1 derivative of the inventionhas a potency (EC₅₀ in nM), as determined using the cAMP assay, below0.090, preferably below 0.080, more preferably below 0.070, even morepreferably below 0.060, and most preferably below 0.050 (nM).

In a fifth particular embodiment, the GLP-1 derivative of the inventionhas a potency (EC₅₀ in nM), as determined using the cAMP assay, below0.040, preferably below 0.030, more preferably below 0.020, and mostpreferably below 0.010 (nM).

Accordingly, exemplary ranges of potency (EC₅₀ in nM, as determinedusing the cAMP assay) of GLP-1 derivatives of the invention are0.010-2.00, 0.010-1.80, 0.010-1.60, 0.010-1.40, 0.010-1.20, 0.010-1.00,0.010-0.80, 0.010-0.60, 0.010-0.40, 0.010-0.30, 0.010-0.20, 0.010-0.10,and 0.010-0.90 (nM), preferably 0.010-0.40, 0.010-0.30, 0.010-0.20,0.010-0.10, and 0.010-0.90 (nM).

In a sixth particular embodiment, for very strong albumin bindinganalogues with albumin binding affinity below 100 nM, the GLP-1 potencyis better than 3 micro molar and preferable the potency is better than 1micromolar in the cAMP assay. For strong albumin binding derivativeswith albumin binding affinity below 500 nM, the GLP-1 potency is betterthan 1 micro molar and preferable the potency is better than 0.2micromolar in the cAMP assay.

In a seventh particular embodiment, the GLP-1 derivative of theinvention can bind to albumin and the GLP-1 receptor simultaneously. Forexample, the GLP-1 derivative of the invention may bind to the GLP-1receptor with an affinity below 100 nM, preferable below 30 nM in thepresence of 2% albumin.

The GLP-1 derivative of the invention may also have an affinity to theGLP-1 receptor which is only partly decreased when comparing theaffinity in the presence of very low concentration (e.g. 0.005% to 0.2%)of human albumin to the affinity in the presence of 2% human albumin.The shift in binding affinity under these conditions is preferably lessthan 50 fold, more preferably below 30 fold and most preferably below 10fold.

In a fourth aspect, the GLP-1 derivative of the invention has a highalbumin binding affinity. The albumin refers to human serum albumin(HSA), and the affinity may be determined as described in Example 41.

The affinities of the GLP-1 derivatives for human serum albumin (HSA)may be measured by a competition scintillation proximity assay (SPA),preferably by (i) incubating streptavidin-SPA beads (such as GEHealthcare RPNQ0009) with biotinylated HSA, e.g. for 5 hours; (ii)washing the beads with buffer; (iii) mixing the beads mixed with an¹²⁵I-labeled acylated GLP-1 analogue, such asN-epsilon26-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,¹²⁵I-Tyr19,Arg34]GLP-1(7-37)orN-epsilon37-[2-(2-[2-((S)-4-((S)-4-(12-[4-(16-(1H-tetrazol-5-yl)hexadecanoylsulfamoyl)butyrylamino]dodecanoylamino)-4-carboxybutyrylamino)-4-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Aib8,¹²⁵I-Tyr19,Glu22,Arg26,34,Lys37]GLP-1(7-37)-NH₂, preferably in a buffer containing 100 mM Hepes, 100 mMNaCl, 10 mM MgSO₄, 0.025% Tween-20, pH 7.4; (iv) transferring themixture into the wells of a scintillation counter such as Perkin ElmerOptiplate-96 6005290 (100 μl per well), using suitable volumes such as100 μl of a suitable dilution series of the GLP-1 derivative to bemeasured, preferably in the same buffer; (v) centrifuging the platesafter a suitable incubation time such as 20 hours, preferably withgentle rocking, more preferably at room temperature; (vi) counting theplates, e.g. on a TopCounter; and/or (vii) plotting bound cpm as afunction of GLP-1 derivative concentration. The EC₅₀ value of thecompetition curve is preferably used as a measure of the affinity of thederivative for HSA. The HSA binding affinity may also be expressed asK_(d) apparent (K_(d) for Dissociation Equilibrium Constant).

In a first particular embodiment, the albumin binding affinity (i.e. theEC₅₀ value (in nM) of the competition curve, as measured using the assayof Example 41), is below 2000, preferably below 1500, more preferablybelow 1000, even more preferably below 800, and most preferably below600 (nM).

In a second particular embodiment, the albumin binding affinity (i.e.the EC₅₀ value (in nM) of the competition curve, as measured using theassay of Example 41), is below 500, preferably below 400, morepreferably below 300, even more preferably below 200, and mostpreferably below 100 (nM).

In a third particular embodiment, the albumin binding affinity of theGLP-1 derivative of the invention is below 1 micromolar, preferablybelow 500 nM, and even more preferably below 200 nM, or even below 100nM.

Accordingly, exemplary ranges of albumin binding affinity (EC50 in nM)of the GLP-1 derivative of the invention are: 1-2000, 100-2000,200-2000, 400-1500, 600-1500, and 800-1500 (nM).

In a fifth aspect, the GLP-1 derivative of the invention has a highaffinity binding to the isolated N-terminal extracellular domain of theGLP-1 receptor (nGLP-1R). The affinity may be measured as the ability todisplace ¹²⁵I-Exendin-4(9-39) from binding to nGLP-1R, e.g. as describedin Example 42.

The protein nGLP-1R may be prepared as described by Runge et al 2007 (InBiochemistry, vol. 46, pp. 5830-5840). The protein is then biotinylatedand immobilized, preferably on streptavidin-coated SPA beads. The nGLP1Rin a suitable buffer such as 0.1M NaHCO₃ may be biotinylated using 75 μgBNHS (Sigma H1759) to 1 mg protein. The biotinylated nGLP1R issubsequently preferably dialyzed against PBS. All reagents andderivatives are preferably diluted in PBS, preferably with 0.05% v/vTween 20. The binding assay may e.g. be carried out in 96 wellOptiPlates (PerkinElmer 6005290) in a final volume of 200 μl. Each wellmay contain 2 mg streptavidin coated SPA beads (such as PerkinElmerRPNQ007), 0.1 pmol biotinylated nGLP1R, 50 pCi ¹²⁵I-Exendin (9-39) andtest peptide in suitable final concentrations, e.g. ranging from 1000 nMto 0.064 nM. The plates are incubated on a shaker, preferably at RT(room temperature), or at 20° C., and for a suitable period of time,e.g. 3 hours. The SPA particles may be spun down by centrifugation, e.g.for 10 min at 1500 rpm, and the plates are counted, e.g. in aTopCount-NXT (PerkinElmer).

The affinity may be expressed by way of an IC₅₀ value, which is readfrom the curve as the concentration of the GLP-1 derivative whichdisplaces 50% of ¹²⁵I-Exendin-4(9-39) from binding to nGLP-1R.

In a first particular embodiment, the GLP-1 derivative of the inventionhas an affinity to the extracellular domain of the GLP-1 receptor(nGLP-1R), measured as IC₅₀/nM in the assay of Example 42, of below1500, preferably below 1000, even more preferably below 500, and mostpreferably below 400 (nM).

In a second particular embodiment, the GLP-1 derivative of the inventionhas an affinity to the extracellular domain of the GLP-1 receptor(nGLP-1R), measured as IC₅₀/nM in the assay of Example 42, of below 300,preferably below 200, even more preferably below 150, and mostpreferably below 100 (nM).

In a third particular embodiment, the GLP-1 derivative of the inventionhas an affinity to the extracellular domain of the GLP-1 receptor(nGLP-1R), measured as IC₅₀/nM in the assay of Example 42, of below 80,preferably below 60, even more preferably below 40, and most preferablybelow 20 (nM).

In a fourth particular embodiment, the GLP-1 derivative of the inventionhas an affinity to the extracellular domain of the GLP-1 receptor(nGLP-1R), measured as IC₅₀/nM in the assay of Example 42, of below 15,preferably below 10, even more preferably below 8, and most preferablybelow 6 (nM).

In a fifth particular embodiment, the GLP-1 derivative of the inventionhas an affinity to the extracellular domain of the GLP-1 receptor(nGLP-1R), measured as IC₅₀/nM in the assay of Example 42, of below 5.0,preferably below 4.0, even more preferably below 3.0, and mostpreferably below 2.0 (nM).

Accordingly, exemplary ranges of affinity to nGLP-1R (IC₅₀ in nM) of theGLP-1 derivative of the invention are: 2-1500, 2-1000, 2-500, 2-300,5-500, 10-500, and 2-10 (nM).

In this assay Exendin-4 binds nGLP-1R with an IC₅₀ value of 5 nM,GLP-1(7-37) binds nGLP-1R with an IC₅₀ value of 1120 nM and liraglutidebinds nGLP-1R with an IC₅₀ value of 1500 nM.

In a sixth particular embodiment, the GLP-1 derivative of the inventionbinds nGLP-1R with an IC₅₀ value lower than that of liraglutide, morepreferably with an IC₅₀ value lower than 100 nM, and even morepreferably below 10 nM, or even below 5 nM.

In a sixth aspect, The term “DPP-IV protected” as used herein referringto a polypeptide means a polypeptide which has been chemically modifiedin order to render said derivative resistant to the plasma peptidasedipeptidyl aminopeptidase-4 (DPP-IV). The DPP-IV enzyme in plasma isknown to be involved in the degradation of several peptide hormones,e.g. GLP-1, GLP-2, Exendin-4 etc. Thus, a considerable effort is beingmade to develop analogues and derivatives of the polypeptidessusceptible to DPP-IV mediated hydrolysis in order to reduce the rate ofdegradation by DPP-IV.

In one embodiment a derivative according to the invention is a DPP-IVprotected derivative which is more resistant to DPP-IV than liraglutide.

Resistance of a peptide to degradation by dipeptidyl aminopeptidase IVis determined by the following degradation assay: Aliquots of thepeptide (5 nmol) are incubated at 37° C. with 1 μL of purifieddipeptidyl aminopeptidase IV corresponding to an enzymatic activity of 5mU for 10-180 minutes in 100 μL of 0.1 M triethylamine-HCl buffer, pH7.4. Enzymatic reactions are terminated by the addition of 5 μL of 10%trifluoroacetic acid, and the peptide degradation products are separatedand quantified using HPLC analysis. One method for performing thisanalysis is: The mixtures are applied onto a Vydac C18 widepore (30 nmpores, 5 μm particles) 250×4.6 mm column and eluted at a flow rate of 1ml/min with linear stepwise gradients of acetonitrile in 0.1%trifluoroacetic acid (0% acetonitrile for 3 min, 0-24% acetonitrile for17 min, 24-48% acetonitrile for 1 min) according to Siegel et al.,Regul. Pept. 1999; 79:93-102 and Mentlein et al. Eur. J. Biochem. 1993;214:829-35. Peptides and their degradation products may be monitored bytheir absorbance at 220 nm (peptide bonds) or 280 nm (aromatic aminoacids), and are quantified by integration of their peak areas related tothose of standards. The rate of hydrolysis of a peptide by dipeptidylaminopeptidase IV is estimated at incubation times which result in lessthan 10% of the peptide being hydrolysed.

Alternatively, the resistance of a peptide to degradation by dipeptidylaminopeptidase IV is determined by the following degradation assay:Aliquots of the peptide (4 nmol) are incubated at 37° C. with 10.9 mU ofpurified dipeptidyl aminopeptidase IV for 22 hours in 40 μL of 0.085 MTris-HCl buffer, pH 8.0, in presence or absence of 1.6% human serumalbumin. After 0, 4, and 22 hours samples of 10 μl are taken andenzymatic reactions are terminated by mixing with 100 μl of 1%trifluoroacetic acid. The peptide degradation products are separated andquantified using HPLC analysis. One method for performing this analysisis: The mixtures are applied onto an Agilent Zorbax 300SB-C18 (5 μmparticles) 150×2.1 mm column and eluted at a flow rate of 0.5 ml/minwith a linear gradient from 0.1% trifluoroacetic acid to 100%acetonitrile with 0.07% TFA in 30 minutes. Peptides and theirdegradation products are monitored by their absorbance at 214 nm, andare quantified by integration of their peak areas. The stability of apeptide against dipeptidyl aminopeptidase IV is determined as the peakarea of the intact peptide relative to the sum of the peak areas of theintact peptide and the degradation product lacking the two aminoterminalamino acids after cleavage.

In a seventh aspect, the present invention relates to a derivative of aGLP-1 peptide which can be formulated into particles suitable forpulmonary administration (delivery). This may be with regard to physicalor chemical aspects which are useful for a pulmonal formulation.Alternatively, the derivatives are stable against degradation by enzymesin the airways and lungs.

In embodiments of the invention a combination of one or more of theabove features is achieved.

Albumin Binding

The term “albumin binding moiety” as used herein means a residue whichbinds non-covalently to human serum albumin. The albumin binding residueattached to the therapeutic polypeptide typically has an albumin bindingaffinity that is below 1 micromolar, preferable below 500 nM and evenmore preferable below 200 nM or even below 100 nM.

A range of albumin binding residues are known among linear and branchedlipohophillic moieties containing 4-40 carbon atoms having a distalacidic group.

The term “hydrophilic linker” as used herein means a spacer thatseparates a peptide and an albumin binding residue with a chemicalmoiety which comprises at least 5 non-hydrogen atoms where 30-50% ofthese are either N or O.

In the formulas below the terminal bonds from the attached groups are tobe regarded as attachment bonds and not ending in methylene groupsunless stated.

Another object of the present invention is to provide a pharmaceuticalformulation comprising a derivative according to the present inventionwhich is present in a concentration from 0.1 mg/ml to 25 mg/ml, andwherein said formulation has a pH from 3.0 to 9.0. The formulation mayfurther comprise a buffer system, preservative(s), tonicity agent(s),chelating agent(s), stabilizers and surfactants.

Formulation

In one embodiment of the invention, the pharmaceutical formulation is anaqueous formulation, i.e. formulation comprising water. Such formulationis typically a solution or a suspension.

In a further embodiment of the invention, the pharmaceutical formulationis an aqueous solution.

The term “aqueous formulation” is defined as a formulation comprising atleast 50% w/w water. Likewise, the term “aqueous solution” is defined asa solution comprising at least 50% w/w water, and the term “aqueoussuspension” is defined as a suspension comprising at least 50% w/wwater.

In another embodiment, the pharmaceutical formulation is a freeze-driedformulation, whereto the physician or the patient adds solvents and/ordiluents prior to use.

In another embodiment, the pharmaceutical formulation is a driedformulation (e.g. freeze-dried or spray-dried) ready for use without anyprior dissolution.

In a further aspect, the invention relates to a pharmaceuticalformulation comprising an aqueous solution of a derivative according tothe present invention, and a buffer, wherein said derivative is presentin a concentration from 0.1 mg/ml or above, and wherein said formulationhas a pH from about 3.0 to about 9.0.

In another embodiment of the invention, the pH of the formulation isfrom about 7.0 to about 9.5. In another embodiment of the invention, thepH of the formulation is from about 3.0 to about 7.0. In anotherembodiment of the invention, the pH of the formulation is from about 5.0to about 7.5. In another embodiment of the invention, the pH of theformulation is from about 7.5 to about 9.0. In another embodiment of theinvention, the pH of the formulation is from about 7.5 to about 8.5. Inanother embodiment of the invention, the pH of the formulation is fromabout 6.0 to about 7.5. In another embodiment of the invention, the pHof the formulation is from about 6.0 to about 7.0. In anotherembodiment, the pharmaceutical formulation is from 8.0 to 8.5.

In an embodiment of the invention, each administered dose contains from0.01 mg-10 mg of active derivative. In an embodiment, the doseadministered contains more than 0.05 mg active derivative. In anembodiment, the dose administered contains more than 0.1 mg activederivative. In an embodiment, the dose administered contains up to 10 mgactive derivative. In an embodiment, the dose administered contains upto 9 mg active derivative. In an embodiment, the dose administeredcontains up to 8 mg active derivative. In an embodiment, the doseadministered contains up to 7 mg active derivative. In an embodiment,the dose administered contains up to 6 mg active derivative. In anembodiment, the dose administered contains up to 5 mg active derivative.In an embodiment, the dose administered contains from 0.2 mg to 5 mgactive derivative.

In a further embodiment of the invention, the buffer is selected fromthe group consisting of sodium acetate, sodium carbonate, citrate,glycylglycine, histidine, glycine, lysine, arginine, sodium dihydrogenphosphate, disodium hydrogen phosphate, sodium phosphate, andtris(hydroxymethyl)-aminomethan, bicine, tricine, malic acid, succinate,maleic acid, fumaric acid, tartaric acid, aspartic acid or mixturesthereof. Each one of these specific buffers constitutes an alternativeembodiment of the invention.

In a further embodiment of the invention, the formulation furthercomprises a pharmaceutically acceptable preservative. In a furtherembodiment of the invention the preservative is selected from the groupconsisting of phenol, o-cresol, m-cresol, p-cresol, methylp-hydroxybenzoate, propyl p-hydroxybenzoate, 2-phenoxyethanol, butylp-hydroxybenzoate, 2-phenylethanol, benzyl alcohol, chlorobutanol, andthiomersal, bronopol, benzoic acid, imidurea, chlorohexidine, sodiumdehydroacetate, chlorocresol, ethyl p-hydroxybenzoate, benzethoniumchloride, chlorphenesine (3p-chlorophenoxypropane-1,2-diol) or mixturesthereof. In an embodiment, the preservative is phenol or m-cresol. In afurther embodiment of the invention, the preservative is present in aconcentration from 0.1 mg/ml to 20 mg/ml. In a further embodiment of theinvention, the preservative is present in a concentration from 0.1 mg/mlto 5 mg/ml. In a further embodiment of the invention, the preservativeis present in a concentration from 5 mg/ml to 10 mg/ml. In a furtherembodiment of the invention, the preservative is present in aconcentration from 10 mg/ml to 20 mg/ml. Each one of these specificpreservatives constitutes an alternative embodiment of the invention.The use of a preservative in pharmaceutical compositions is well-knownto the skilled person. For convenience reference is made to Remington:The Science and Practice of Pharmacy, 19^(th) edition, 1995.

In a further embodiment of the invention, the formulation furthercomprises an isotonic agent. In a further embodiment of the invention,the isotonic agent is selected from the group consisting of a salt (e.g.sodium chloride), a sugar or sugar alcohol, an amino acid (e.g.L-glycine, L-histidine, arginine, lysine, isoleucine, aspartic acid,tryptophan, threonine), an alditol (e.g. glycerol (glycerine),1,2-propanediol (propyleneglycol), 1,3-propanediol, 1,3-butanediol)polyethyleneglycol (e.g. PEG400), or mixtures thereof. In an embodiment,the isotonicity agent is propyleneglycol. Any sugar such as mono-, di-,or polysaccharides, or water-soluble glucans, including for examplefructose, glucose, mannose, sorbose, xylose, maltose, lactose, sucrose,trehalose, dextran, pullulan, dextrin, cyclodextrin, alfa and beta HPCD,soluble starch, hydroxyethyl starch and carboxymethylcellulose-Na may beused. In one embodiment, the sugar additive is sucrose. Sugar alcohol isdefined as a C4-C8 hydrocarbon having at least one —OH group andincludes, for example, mannitol, sorbitol, inositol, galactitol,dulcitol, xylitol, and arabitol. In one embodiment, the sugar alcoholadditive is mannitol. The sugars or sugar alcohols mentioned above maybe used individually or in combination. There is no fixed limit to theamount used, as long as the sugar or sugar alcohol is soluble in theliquid preparation and does not adversely effect the stabilizing effectsachieved using the methods of the invention. In one embodiment, thesugar or sugar alcohol concentration is between about 1 mg/ml and about150 mg/ml. In a further embodiment of the invention, the isotonic agentis present in a concentration from 1 mg/ml to 50 mg/ml. In a furtherembodiment of the invention, the isotonic agent is present in aconcentration from 1 mg/ml to 7 mg/ml. In an embodiment of theinvention, the isotonic agent is present in a concentration from 5 mg/mlto 7 mg/ml. In a further embodiment of the invention, the isotonic agentis present in a concentration from 8 mg/ml to 24 mg/ml. In a furtherembodiment of the invention, the isotonic agent is present in aconcentration from 25 mg/ml to 50 mg/ml. Each one of these specificisotonic agents constitutes an alternative embodiment of the invention.The use of an isotonic agent in pharmaceutical compositions iswell-known to the skilled person. For convenience reference is made toRemington: The Science and Practice of Pharmacy, 19^(th) edition, 1995.

In a further embodiment of the invention, the formulation furthercomprises a chelating agent. In a further embodiment of the inventionthe chelating agent is selected from salts of ethylenediaminetetraaceticacid (EDTA), citric acid, and aspartic acid, and mixtures thereof. In afurther embodiment of the invention the chelating agent is present in aconcentration from 0.1 mg/ml to 5 mg/ml. In a further embodiment of theinvention the chelating agent is present in a concentration from 0.1mg/ml to 2 mg/ml. In a further embodiment of the invention the chelatingagent is present in a concentration from 2 mg/ml to 5 mg/ml. Each one ofthese specific chelating agents constitutes an alternative embodiment ofthe invention. The use of a chelating agent in pharmaceuticalcompositions is well-known to the skilled person. For conveniencereference is made to Remington: The Science and Practice of Pharmacy,19^(th) edition, 1995.

In a further embodiment of the invention, the formulation furthercomprises a stabilizer. The use of a stabilizer in pharmaceuticalcompositions is well-known to the skilled person. For conveniencereference is made to Remington: The Science and Practice of Pharmacy,19^(th) edition, 1995.

More particularly, compositions of the invention are stabilized liquidpharmaceutical compositions whose therapeutically active componentsinclude a polypeptide that possibly exhibits aggregate formation duringstorage in liquid pharmaceutical formulations.

By “aggregate formation” is intended a physical interaction between thepolypeptide molecules that results in formation of oligomers, which mayremain soluble, or large visible aggregates that precipitate from thesolution. By “during storage” is intended a liquid pharmaceuticalcomposition or formulation once prepared, is not immediatelyadministered to a subject. Rather, following preparation, it is packagedfor storage, either in a liquid form, in a frozen state, or in a driedform for later reconstitution into a liquid form or other form suitablefor administration to a subject. By “dried form” is intended the liquidpharmaceutical composition or formulation is dried either by freezedrying (i.e., lyophilization; see, for example, Williams and Polli(1984) J. Parenteral Sci. Technol. 38:48-59), spray drying (see Masters(1991) in Spray-Drying Handbook (5th ed; Longman Scientific andTechnical, Essez, U.K.), pp. 491-676; Broadhead et al. (1992) DrugDevel. Ind. Pharm. 18:1169-1206; and Mumenthaler et al. (1994) Pharm.Res. 11:12-20), or air drying (Carpenter and Crowe (1988) Cryobiology25:459-470; and Roser (1991) Biopharm. 4:47-53). Aggregate formation bya polypeptide during storage of a liquid pharmaceutical composition canadversely affect biological activity of that polypeptide, resulting inloss of therapeutic efficacy of the pharmaceutical composition.Furthermore, aggregate formation may cause other problems such asblockage of tubing, membranes, or pumps when the polypeptide-containingpharmaceutical composition is administered using an infusion system.

The pharmaceutical compositions of the invention may further comprise anamount of an amino acid base sufficient to decrease aggregate formationby the polypeptide during storage of the composition. By “amino acidbase” is intended an amino acid or a combination of amino acids, whereany given amino acid is present either in its free base form or in itssalt form. Where a combination of amino acids is used, all of the aminoacids may be present in their free base forms, all may be present intheir salt forms, or some may be present in their free base forms whileothers are present in their salt forms. In one embodiment, amino acidsto use in preparing the compositions of the invention are those carryinga charged side chain, such as arginine, lysine, aspartic acid, andglutamic acid. Any stereoisomer (i.e., L, D, or a mixture thereof) of aparticular amino acid (e.g. methionine, histidine, imidazole, arginine,lysine, isoleucine, aspartic acid, tryptophan, threonine and mixturesthereof) or combinations of these stereoisomers, may be present in thepharmaceutical compositions of the invention so long as the particularamino acid is present either in its free base form or its salt form.

In one embodiment the L-stereoisomer is used. Compositions of theinvention may also be formulated with analogues of these amino acids. By“amino acid analogue” is intended a derivative of the naturallyoccurring amino acid that brings about the desired effect of decreasingaggregate formation by the polypeptide during storage of the liquidpharmaceutical compositions of the invention. Suitable arginineanalogues include, for example, aminoguanidine, ornithine andN-monoethyl L-arginine, suitable methionine analogues include ethionineand buthionine and suitable cysteine analogues include S-methyl-Lcysteine. As with the other amino acids, the amino acid analogues areincorporated into the compositions in either their free base form ortheir salt form. In a further embodiment of the invention the aminoacids or amino acid analogues are used in a concentration, which issufficient to prevent or delay aggregation of the protein.

In a further embodiment of the invention, methionine (or other sulphuricamino acids or amino acid analogous) may be added to inhibit oxidationof methionine residues to methionine sulfoxide when the polypeptideacting as the therapeutic agent is a polypeptide comprising at least onemethionine residue susceptible to such oxidation. By “inhibit” isintended minimal accumulation of methionine oxidized species over time.Inhibiting methionine oxidation results in greater retention of thepolypeptide in its proper molecular form. Any stereoisomer of methionine(L or D) or combinations thereof can be used. The amount to be addedshould be an amount sufficient to inhibit oxidation of the methionineresidues such that the amount of methionine sulfoxide is acceptable toregulatory agencies. Typically, this means that the composition containsno more than about 10% to about 30% methionine sulfoxide. Generally,this can be achieved by adding methionine such that the ratio ofmethionine added to methionine residues ranges from about 1:1 to about1000:1, such as 10:1 to about 100:1.

In a further embodiment of the invention, the formulation furthercomprises a stabilizer selected from the group of high molecular weightpolymers or low molecular compounds.

In a further embodiment of the invention the stabilizer is selected frompolyethylene glycol (e.g. PEG 3350), polyvinyl alcohol (PVA),polyvinylpyrrolidone, carboxy-/hydroxycellulose or derivates thereof(e.g. HPC, HPC-SL, HPC-L and HPMC), cyclodextrins, sulphur-containingsubstances as monothioglycerol, thioglycolic acid and2-methylthioethanol, and different salts (e.g. sodium chloride). Eachone of these specific stabilizers constitutes an alternative embodimentof the invention.

The pharmaceutical compositions may also comprise additional stabilizingagents, which further enhance stability of a therapeutically activepolypeptide therein.

Stabilizing agents of particular interest to the present inventioninclude, but are not limited to, methionine and EDTA, which protect thepolypeptide against methionine oxidation, and a nonionic surfactant,which protects the polypeptide against aggregation associated withfreeze-thawing or mechanical shearing.

In a further embodiment of the invention, the formulation furthercomprises a surfactant. In another embodiment of the invention, thepharmaceutical composition comprises two different surfactants. The term“Surfactant” as used herein refers to any molecules or ions that arecomprised of a water-soluble (hydrophilic) part, the head, and afat-soluble (lipophilic) segment. Surfactants accumulate preferably atinterfaces, which the hydrophilic part is orientated towards the water(hydrophilic phase) and the lipophilic part towards the oil- orhydrophobic phase (i.e. glass, air, oil etc.). The concentration atwhich surfactants begin to form micelles is known as the criticalmicelle concentration or CMC. Furthermore, surfactants lower the surfacetension of a liquid. Surfactants are also known as amphipathiccompounds. The term “Detergent” is a synonym used for surfactants ingeneral.

Anionic surfactants may be selected from the group of: Chenodeoxycholicacid, Chenodeoxycholic acid sodium salt, Cholic acid, Dehydrocholicacid, Deoxycholic acid, Deoxycholic acid methyl ester, Digitonin,Digitoxigenin, N,N-Dimethyldodecylamine N-oxide, Docusate sodium,Glycochenodeoxycholic acid sodium, Glycocholic acid hydrate,Glycodeoxycholic acid monohydrate, Glycodeoxycholic acid sodium salt,Glycodeoxycholic acid sodium salt, Glycolithocholic acid 3-sulfatedisodium salt, Glycolithocholic acid ethyl ester, N-Lauroylsarcosinesodium salt, N-Lauroylsarcosine sodium salt, N-Lauroylsarcosine,N-Lauroylsarcosine, Lithium dodecyl sulfate, Lugol, 1-Octanesulfonicacid sodium salt, 1-Octanesulfonic acid sodium salt, Sodium1-butanesulfonate, Sodium 1-decanesulfonate, Sodium 1-dodecanesulfonate,Sodium 1-heptanesulfonate, Sodium 1-heptanesulfonate, Sodium1-nonanesulfonate, Sodium 1-propanesulfonate monohydrate, Sodium2-bromoethanesulfonate, Sodium cholate hydrate, ox or sheep bile, Sodiumcholate hydrate, Sodium cholate, Sodium deoxycholate, Sodium dodecylsulfate, Sodium dodecyl sulfate, Sodium hexanesulfonate, Sodium octylsulfate, Sodium pentanesulfonate, Sodium taurocholate,Taurochenodeoxycholic acid sodium salt, Taurodeoxycholic acid sodiumsalt monohydrate, Taurolithocholic acid 3-sulfate disodium salt,Tauroursodeoxycholic acid sodium salt, Trizma® dodecyl sulfate, DSS(docusate sodium, CAS registry no [577-11-7]), docusate calcium, CASregistry no [128-49-4]), docusate potassium, CAS registry no[7491-09-0]), SDS (sodium dodecyl sulfate or sodium lauryl sulfate),Dodecylphosphocholine (FOS-Choline-12), Decylphosphocholine(FOS-Choline-10), Nonylphosphocholine (FOS-Choline-9), dipalmitoylphosphatidic acid, sodium caprylate, and/or Ursodeoxycholic acid.

Cationic surfactants may be selected from the group of:Alkyltrimethylammonium bromide Benzalkonium chloride, Benzalkoniumchloride, Benzyldimethylhexadecylammonium chloride,Benzyldimethyltetradecylammonium chloride, Benzyltrimethylammoniumtetrachloroiodate, Dimethyldioctadecylammonium bromide,Dodecylethyldimethylammonium bromide, Dodecyltrimethylammonium bromide,Dodecyltrimethylammonium bromide, Ethylhexadecyldimethylammoniumbromide, Hexadecyltrimethylammonium bromide, Hexadecyltrimethylammoniumbromide, Polyoxyethylene(10)-N-tallow-1,3-diaminopropane, Thonzoniumbromide, and/or Trimethyl(tetradecyl)ammonium bromide.

Nonionic surfactants may be selected from the group of: BigCHAP,Bis(polyethylene glycol bis[imidazoyl carbonyl]), block copolymers as

polyethyleneoxide/polypropyleneoxide block copolymers such aspoloxamers, poloxamer 188 and poloxamer 407, Brij® 35, Brij® 56, Brij®72, Brij® 76, Brij® 92V, Brij® 97, Brij® 58P, Cremophor® EL,Decaethylene glycol monododecyl ether, N-Decanoyl-N-methylglucamine,n-Dodecanoyl-N-methylglucamide, alkyl-polyglucosides, ethoxylated castoroil, Heptaethylene glycol monodecyl ether, Heptaethylene glycolmonododecyl ether, Heptaethylene glycol monotetradecyl ether,Hexaethylene glycol monododecyl ether, Hexaethylene glycol monohexadecylether, Hexaethylene glycol monooctadecyl ether, Hexaethylene glycolmonotetradecyl ether, Igepal CA-630, Igepal CA-630,Methyl-6-O—(N-heptylcarbamoyl)-beta-D-glucopyranoside, Nonaethyleneglycol monododecyl ether, N-Nonanoyl-N-methylglucamine,N-Nonanoyl-N-methylglucamine, Octaethylene glycol monodecyl ether,Octaethylene glycol monododecyl ether, Octaethylene glycol monohexadecylether, Octaethylene glycol monooctadecyl ether, Octaethylene glycolmonotetradecyl ether, Octyl-β-D-glucopyranoside, Pentaethylene glycolmonodecyl ether, Pentaethylene glycol monododecyl ether, Pentaethyleneglycol monohexadecyl ether, Pentaethylene glycol monohexyl ether,Pentaethylene glycol monooctadecyl ether, Pentaethylene glycol monooctylether, Polyethylene glycol diglycidyl ether, Polyethylene glycol etherW-1, Polyoxyethylene 10 tridecyl ether, Polyoxyethylene 100 stearate,Polyoxyethylene 20 isohexadecyl ether, Polyoxyethylene 20 oleyl ether,Polyoxyethylene 40 stearate, Polyoxyethylene 50 stearate,Polyoxyethylene 8 stearate, Polyoxyethylene bis(imidazolyl carbonyl),Polyoxyethylene 25 propylene glycol stearate, Saponin from Quillajabark, Span® 20, Span® 40, Span® 60, Span® 65, Span® 80, Span® 85,Tergitol, Type 15-S-12, Tergitol, Type 15-S-30, Tergitol, Type 15-S-5,Tergitol, Type 15-S-7, Tergitol, Type 15-S-9, Tergitol, Type NP-10,Tergitol, Type NP-4, Tergitol, Type NP-40, Tergitol, Type NP-7,Tergitol, Type NP-9, Tetradecyl-β-D-maltoside, Tetraethylene glycolmonodecyl ether, Tetraethylene glycol monododecyl ether, Tetraethyleneglycol monotetradecyl ether, Triethylene glycol monodecyl ether,Triethylene glycol monododecyl ether, Triethylene glycol monohexadecylether, Triethylene glycol monooctyl ether, Triethylene glycolmonotetradecyl ether, Triton CF-21, Triton CF-32, Triton DF-12, TritonDF-16, Triton GR-5M, Triton QS-15, Triton QS-44, Triton X-100, TritonX-102, Triton X-15, Triton X-151, Triton X-200, Triton X-207, Triton®X-100, Triton® X-114, Triton® X-165 solution, Triton® X-305 solution,Triton® X-405, Triton® X-45, Triton® X-705-70, TWEEN® 20, TWEEN® 40,TWEEN® 60, TWEEN® 6, TWEEN® 65, TWEEN® 80, TWEEN® 81, TWEEN® 85,Tyloxapol, sphingophospholipids (sphingomyelin), and sphingoglycolipids(ceramides, gangliosides), phospholipids, and/or n-Undecylβ-D-glucopyranoside.

Zwitterionic surfactants may be selected from the group of: CHAPS,CHAPSO, 3-(Decyldimethylammonio)propanesulfonate inner salt,3-(Dodecyldimethylammonio)-propanesulfonate inner salt,3-(Dodecyldimethylammonio)propanesulfonate inner salt,3-(N,N-Dimethylmyristylammonio)propanesulfonate,3-(N,N-Dimethyloctadecyl-ammonio)propanesulfonate,3-(N,N-Dimethyloctylammonio)propanesulfonate inner salt,3-(N,N-Dimethylpalmitylammonio)propanesulfonate,N-alkyl-N,N-dimethylammonio-1-propanesulfonates,3-cholamido-1-propyldimethylammonio-1-propanesulfonate,Dodecylphosphocholine, myristoyl lysophosphatidylcholine, Zwittergent3-12 (N-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), Zwittergent3-10 (3-(Decyldimethyl-ammonio)propanesulfonate inner salt), Zwittergent3-08 (3-(Octyldimethyl-ammonio)pro-panesulfonate), glycerophospholipids(lecithins, kephalins, phosphatidyl serine), glyceroglycolipids(galactopyranoside), alkyl, alkoxyl (alkyl ester), alkoxy (alkylether)-derivatives of lysophosphatidyl and phosphatidylcholines, e.g.lauroyl and myristoyl derivatives of lysophosphatidylcholine,dipalmitoylphosphatidylcholine, and modifications of the polar headgroup, that is cholines, ethanolamines, phosphatidic acid, serines,threonines, glycerol, inositol, lysophosphatidylserine andlysophosphatidylthreonine, acylcarnitines and derivatives,N^(beta)-acylated derivatives of lysine, arginine or histidine, orside-chain acylated derivatives of lysine or arginine, N^(beta)-acylatedderivatives of dipeptides comprising any combination of lysine, arginineor histidine and a neutral or acidic amino acid, N^(beta)-acylatedderivative of a tripeptide comprising any combination of a neutral aminoacid and two charged amino acids, or the surfactant may be selected fromthe group of imidazoline derivatives, long-chain fatty acids and saltsthereof C₆-C₁₂ (eg. oleic acid and caprylic acid),N-Hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, anionic(alkyl-aryl-sulphonates) monovalent surfactants, palmitoyllysophosphatidyl-L-serine, lysophospholipids (e.g.1-acyl-sn-glycero-3-phosphate esters of ethanolamine, choline, serine orthreonine), or mixtures thereof.

The term “alkyl-polyglucosides” as used herein in relates to an straightor branched C₅₋₂₀-alkyl, -alkenyl or -alkynyl chain which is substitutedby one or more glucoside moieties such as maltoside, saccharide etc.Embodiments of these alkyl-polyglucosides includeC₆₋₁₈-alkyl-polyglucosides. Specific embodiments of thesealkyl-polyglucosides includes the even numbered carbon-chains such asC₆, C₈, C₁₀, C₁₂, C₁₄, C₁₆, C₁₈ and C₂₀ alkyl chain. Specificembodiments of the glucoside moieties include pyranoside,glucopyranoside, maltoside, maltotrioside and sucrose. In embodiments ofthe invention, less than 6 glucosid moieties are attached to the alkylgroup. In embodiments of the invention, less than 5 glucosid moietiesare attached to the alkyl group. In embodiments of the invention, lessthan 4 glucosid moieties are attached to the alkyl group. In embodimentsof the invention, less than 3 glucosid moieties are attached to thealkyl group. In embodiments of the invention, less than 2 glucosidmoieties are attached to the alkyl group. Specific embodiments ofalkyl-polyglucosides are alkyl glucosides such n-decylβ-D-glucopyranoside, decyl β-D-maltopyranoside, dodecylβ-D-glucopyranoside, n-dodecyl β-D-maltoside, n-dodecyl β-D-maltoside,n-dodecyl β-D-maltoside, tetradecyl β-D-glucopyranoside, decylβ-D-maltoside, hexadecyl β-D-maltoside, decyl β-D-maltotrioside, dodecylβ-D-maltotrioside, tetradecyl β-D-maltotrioside, hexadecylβ-D-maltotrioside, n-dodecyl-sucrose, n-decyl-sucrose, sucrosemonocaprate, sucrose monolaurate, sucrose monomyristate, and sucrosemonopalmitate.

The use of a surfactant in pharmaceutical compositions is well-known tothe skilled person. For convenience reference is made to Remington: TheScience and Practice of Pharmacy, 19^(th) edition, 1995.

In a further embodiment of the invention, the formulation furthercomprises protease inhibitors such as EDTA (ethylenediamine tetraaceticacid) and benzamidineHCl, but other commercially available proteaseinhibitors may also be used. The use of a protease inhibitor isparticular useful in pharmaceutical compositions comprising zymogens ofproteases in order to inhibit autocatalysis.

It is possible that other ingredients may be present in the peptidepharmaceutical formulation of the present invention. Such additionalingredients may include wetting agents, emulsifiers, antioxidants,bulking agents, tonicity modifiers, chelating agents, metal ions,oleaginous vehicles, proteins (e.g., human serum albumin, gelatine orproteins) and a zwitterion (e.g., an amino acid such as betaine,taurine, arginine, glycine, lysine and histidine). Such additionalingredients, of course, should not adversely affect the overallstability of the pharmaceutical formulation of the present invention.

Pharmaceutical compositions containing a derivative according to thepresent invention may be administered to a patient in need of suchtreatment at several sites, for example, at topical sites, for example,skin and mucosal sites, at sites which bypass absorption, for example,administration in an artery, in a vein, in the heart, and at sites whichinvolve absorption, for example, administration in the skin, under theskin, in a muscle or in the abdomen.

Administration of pharmaceutical compositions according to the inventionmay be through several routes of administration, for example, lingual,sublingual, buccal, in the mouth, oral, in the stomach and intestine,nasal, pulmonary, for example, through the bronchioles and alveoli or acombination thereof, epidermal, dermal, transdermal, vaginal, rectal,ocular, for examples through the conjunctiva, uretal, and parenteral topatients in need of such a treatment.

Compositions of the current invention may be administered in severaldosage forms, for example, as solutions, suspensions, emulsions,microemulsions, multiple emulsion, foams, salves, pastes, plasters,ointments, tablets, coated tablets, chewing gum, rinses, capsules, forexample, hard gelatine capsules and soft gelatine capsules,suppositories, rectal capsules, drops, gels, sprays, powder, aerosols,inhalants, eye drops, ophthalmic ointments, ophthalmic rinses, vaginalpessaries, vaginal rings, vaginal ointments, injection solution, in situtransforming solutions, for example in situ gelling, in situ setting, insitu precipitating, in situ crystallization, infusion solution, andimplants. Compositions of the invention may further be compounded in, orattached to, for example through covalent, hydrophobic and electrostaticinteractions, a drug carrier, drug delivery system and advanced drugdelivery system in order to further enhance stability of the derivativeof the present invention, increase bioavailability, increase solubility,decrease adverse effects, achieve chronotherapy well known to thoseskilled in the art, and increase patient compliance or any combinationthereof. Examples of carriers, drug delivery systems and advanced drugdelivery systems include, but are not limited to, polymers, for examplecellulose and derivatives, polysaccharides, for example dextran andderivatives, starch and derivatives, poly(vinyl alcohol), acrylate andmethacrylate polymers, polylactic and polyglycolic acid and blockco-polymers thereof, polyethylene glycols, carrier proteins, for examplealbumin, gels, for example, thermogelling systems, for example blockco-polymeric systems well known to those skilled in the art, micelles,liposomes, microspheres, nanoparticulates, liquid crystals anddispersions thereof, L2 phase and dispersions there of, well known tothose skilled in the art of phase behaviour in lipid-water systems,polymeric micelles, multiple emulsions, self-emulsifying,self-microemulsifying, cyclodextrins and derivatives thereof, anddendrimers.

Compositions of the current invention are useful in the formulation ofsolids, semisolids, powder and solutions for pulmonary administration ofderivatives of the present invention, using, for example a metered doseinhaler, dry powder inhaler and a nebulizer, all being devices wellknown to those skilled in the art.

Compositions of the current invention are specifically useful in theformulation of controlled, sustained, protracting, retarded, and slowrelease drug delivery systems. More specifically, but not limited to,compositions are useful in formulation of parenteral controlled releaseand sustained release systems (both systems leading to a many-foldreduction in number of administrations), well known to those skilled inthe art. Even more preferably, are controlled release and sustainedrelease systems administered subcutaneous. Without limiting the scope ofthe invention, examples of useful controlled release system andcompositions are hydrogels, oleaginous gels, liquid crystals, polymericmicelles, microspheres, nanoparticles,

Methods to produce controlled release systems useful for compositions ofthe current invention include, but are not limited to, crystallization,condensation, co-crystallization, precipitation, co-precipitation,emulsification, dispersion, high pressure homogenisation, encapsulation,spray drying, microencapsulating, coacervation, phase separation,solvent evaporation to produce microspheres, extrusion and supercriticalfluid processes. General reference is made to Handbook of PharmaceuticalControlled Release (Wise, D. L., ed. Marcel Dekker, New York, 2000) andDrug and the Pharmaceutical Sciences vol. 99: Protein Formulation andDelivery (MacNally, E. J., ed. Marcel Dekker, New York, 2000).

Parenteral administration may be performed by subcutaneous,intramuscular, intraperitoneal or intravenous injection by means of asyringe, optionally a pen-like syringe. Alternatively, parenteraladministration can be performed by means of an infusion pump. A furtheroption is a composition which may be a solution or suspension or apowder for the administration of the derivative of the present inventionin the form of a nasal or pulmonal liquid or powder spray. As a stillfurther option, the pharmaceutical compositions containing thederivative of the invention can also be adapted to transdermaladministration, e.g. by needle-free injection or from a patch,optionally an iontophoretic patch, or transmucosal, e.g. buccal,administration.

The derivatives of the present invention can be administered via thepulmonary route in a vehicle, as a solution, suspension or dry powderusing any of known types of devices suitable for pulmonary drugdelivery. Examples of these comprise, but are not limited to, the threegeneral types of aerosol-generating for pulmonary drug delivery, and mayinclude jet or ultrasonic nebulizers, metered-dose inhalers, or drypowder inhalers (Cf. Yu J, Chien Y W. Pulmonary drug delivery:Physiologic and mechanistic aspects. Crit. Rev Ther Drug Carr Sys 14(4)(1997) 395-453).

Based on standardised testing methodology, the aerodynamic diameter(d_(a)) of a particle is defined as the geometric equivalent diameter ofa reference standard spherical particle of unit density (1 g/cm³). Inthe simplest case, for spherical particles, d_(a) is related to areference diameter (d) as a function of the square root of the densityratio as described by:

Modifications to this relationship occur for non-spherical particles(cf. Edwards D A, Ben-Jebria A, Langer R. Recent advances in pulmonarydrug delivery using large, porous inhaled particles. J Appl Physiol84(2) (1998) 379-385). The terms “MMAD” and “MMEAD” are well-describedand known to the art (cf. Edwards D A, Ben-Jebria A, Langer R andrepresents a measure of the median value of an aerodynamic particle sizedistribution. Recent advances in pulmonary drug delivery using large,porous inhaled particles. J Appl Physiol 84(2) (1998) 379-385). Massmedian aerodynamic diameter (MMAD) and mass median effective aerodynamicdiameter (MMEAD) are used inter-changeably, are statistical parameters,and empirically describe the size of aerosol particles in relation totheir potential to deposit in the lungs, independent of actual shape,size, or density (cf. Edwards D A, Ben-Jebria A, Langer R. Recentadvances in pulmonary drug delivery using large, porous inhaledparticles. J Appl Physiol 84(2) (1998) 379-385). MMAD is normallycalculated from the measurement made with impactors, an instrument thatmeasures the particle inertial behaviour in air. In a furtherembodiment, the formulation could be aerosolized by any knownaerosolisation technology, such as nebulisation, to achieve a MMAD ofaerosol particles less than 10 μm, more preferably between 1-5 μm, andmost preferably between 1-3 μm. The preferred particle size is based onthe most effective size for delivery of drug to the deep lung, whereprotein is optimally absorbed (cf. Edwards D A, Ben-Jebria A, Langer A,Recent advances in pulmonary drug delivery using large, porous inhaledparticles. J Appl Physiol 84(2) (1998) 379-385).

Deep lung deposition of the pulmonal formulations comprising thederivative of the present invention may optional be further optimized byusing modifications of the inhalation techniques, for example, but notlimited to: slow inhalation flow (eg. 30 L/min), breath holding andtiming of actuation.

The term “stabilized formulation” refers to a formulation with increasedphysical stability, increased chemical stability or increased physicaland chemical stability.

The term “physical stability” of the protein formulation as used hereinrefers to the tendency of the protein to form biologically inactiveand/or insoluble aggregates of the protein as a result of exposure ofthe protein to thermo-mechanical stresses and/or interaction withinterfaces and surfaces that are destabilizing, such as hydrophobicsurfaces and interfaces. Physical stability of the aqueous proteinformulations is evaluated by means of visual inspection and/or turbiditymeasurements after exposing the formulation filled in suitablecontainers (e.g. cartridges or vials) to mechanical/physical stress(e.g. agitation) at different temperatures for various time periods.Visual inspection of the formulations is performed in a sharp focusedlight with a dark background. The turbidity of the formulation ischaracterized by a visual score ranking the degree of turbidity forinstance on a scale from 0 to 3 (a formulation showing no turbiditycorresponds to a visual score 0, and a formulation showing visualturbidity in daylight corresponds to visual score 3). A formulation isclassified physical unstable with respect to protein aggregation, whenit shows visual turbidity in daylight. Alternatively, the turbidity ofthe formulation can be evaluated by simple turbidity measurementswell-known to the skilled person. Physical stability of the aqueousprotein formulations can also be evaluated by using a spectroscopicagent or probe of the conformational status of the protein. The probe ispreferably a small molecule that preferentially binds to a non-nativeconformer of the protein. One example of a small molecular spectroscopicprobe of protein structure is Thioflavin T. Thioflavin T is afluorescent dye that has been widely used for the detection of amyloidfibrils. In the presence of fibrils, and perhaps other proteinconfigurations as well, Thioflavin T gives rise to a new excitationmaximum at about 450 nm and enhanced emission at about 482 nm when boundto a fibril protein form. Unbound Thioflavin T is essentiallynon-fluorescent at the wavelengths.

Other small molecules can be used as probes of the changes in proteinstructure from native to non-native states. For instance the“hydrophobic patch” probes that bind preferentially to exposedhydrophobic patches of a protein. The hydrophobic patches are generallyburied within the tertiary structure of a protein in its native state,but become exposed as a protein begins to unfold or denature. Examplesof these small molecular, spectroscopic probes are aromatic, hydrophobicdyes, such as antrhacene, acridine, phenanthroline or the like. Otherspectroscopic probes are metal-amino acid complexes, such as cobaltmetal complexes of hydrophobic amino acids, such as phenylalanine,leucine, isoleucine, methionine, and valine, or the like.

The term “chemical stability” of the protein formulation as used hereinrefers to chemical covalent changes in the protein structure leading toformation of chemical degradation products with potential lessbiological potency and/or potential increased immunogenic propertiescompared to the native protein structure. Various chemical degradationproducts can be formed depending on the type and nature of the nativeprotein and the environment to which the protein is exposed. Eliminationof chemical degradation can most probably not be completely avoided andincreasing amounts of chemical degradation products is often seen duringstorage and use of the protein formulation as well-known by the personskilled in the art. Most proteins are prone to deamidation, a process inwhich the side chain amide group in glutaminyl or asparaginyl residuesis hydrolysed to form a free carboxylic acid. Other degradationspathways involves formation of high molecular weight transformationproducts where two or more protein molecules are covalently bound toeach other through transamidation and/or disulfide interactions leadingto formation of covalently bound dimer, oligomer and polymer degradationproducts (Stability of Protein Pharmaceuticals, Ahern. T. J. & ManningM. C., Plenum Press, New York 1992). Oxidation (of for instancemethionine residues) can be mentioned as another variant of chemicaldegradation. The chemical stability of the protein formulation can beevaluated by measuring the amount of the chemical degradation productsat various time-points after exposure to different environmentalconditions (the formation of degradation products can often beaccelerated by for instance increasing temperature). The amount of eachindividual degradation product is often determined by separation of thedegradation products depending on molecule size and/or charge usingvarious chromatography techniques (e.g. SEC-HPLC and/or RP-HPLC).

Hence, as outlined above, a “stabilized formulation” refers to aformulation with increased physical stability, increased chemicalstability or increased physical and chemical stability. In general, aformulation must be stable during use and storage (in compliance withrecommended use and storage conditions) until the expiration date isreached.

In one embodiment of the invention, the pharmaceutical formulationcomprising the derivative of the present invention is stable for morethan 6 weeks of usage and for more than 3 years of storage.

In another embodiment of the invention, the pharmaceutical formulationcomprising the derivative of the present invention is stable for morethan 4 weeks of usage and for more than 3 years of storage.

In a further embodiment of the invention, the pharmaceutical formulationcomprising the derivative of the present invention is stable for morethan 4 weeks of usage and for more than two years of storage.

In an even further embodiment of the invention, the pharmaceuticalformulation comprising the derivative of the present invention is stablefor more than 2 weeks of usage and for more than two years of storage.

In another aspect, the present invention relates to the use of aderivative according to the invention for the preparation of amedicament.

In one embodiment, a derivative according to the invention is used forthe preparation of a medicament for the treatment or prevention ofhyperglycemia, type 2 diabetes, impaired glucose tolerance, type 1diabetes, obesity, hypertension, syndrome X, dyslipidemia, cognitivedisorders, atherosclerosis, myocardial infarction, stroke, coronaryheart disease and other cardiovascular disorders, inflammatory bowelsyndrome, dyspepsia and gastric ulcers.

In another embodiment, a derivative according to the invention is usedfor the preparation of a medicament for delaying or preventing diseaseprogression in type 2 diabetes.

In another embodiment a derivative according to the invention is usedfor the preparation of a medicament for decreasing food intake,decreasing β-cell apoptosis, increasing β-cell function and β-cell mass,and/or for restoring glucose sensitivity to β-cells.

The treatment with a derivative according to the present invention mayalso be combined with a second or more pharmacologically activesubstances, e.g. selected from antidiabetic agents, antiobesity agents,appetite regulating agents, antihypertensive agents, agents for thetreatment and/or prevention of complications resulting from orassociated with diabetes and agents for the treatment and/or preventionof complications and disorders resulting from or associated withobesity. Examples of these pharmacologically active substances are:Insulin, sulphonylureas, biguanides, meglitinides, glucosidaseinhibitors, glucagon antagonists, DPP-IV (dipeptidyl peptidase-IV)inhibitors, inhibitors of hepatic enzymes involved in stimulation ofgluconeogenesis and/or glycogenolysis, glucose uptake modulators,compounds modifying the lipid metabolism such as antihyperlipidemicagents as HMG CoA inhibitors (statins), Gastric Inhibitory Polypeptides(GIP analogs), compounds lowering food intake, RXR agonists and agentsacting on the ATP-dependent potassium channel of the β-cells;Cholestyramine, colestipol, clofibrate, gemfibrozil, lovastatin,pravastatin, simvastatin, probucol, dextrothyroxine, nateglinide,repaglinide; β-blockers such as alprenolol, atenolol, timolol, pindolol,propranolol and metoprolol, ACE (angiotensin converting enzyme)inhibitors such as benazepril, captopril, enalapril, fosinopril,lisinopril, alatriopril, quinapril and ramipril, calcium channelblockers such as nifedipine, felodipine, nicardipine, isradipine,nimodipine, diltiazem and verapamil, and α-blockers such as doxazosin,urapidil, prazosin and terazosin; CART (cocaine amphetamine regulatedtranscript) agonists, NPY (neuropeptide Y) antagonists, PYY agonists, Y2receptor agonists, Y4 receptor agonists, mixed Y2/Y4 receptor agonists,MC4 (melanocortin 4) agonists, orexin antagonists, TNF (tumor necrosisfactor) agonists, CRF (corticotropin releasing factor) agonists, CRF BP(corticotropin releasing factor binding protein) antagonists, urocortinagonists, β3 agonists, oxyntomodulin and analogues, MSH(melanocyte-stimulating hormone) agonists, MCH (melanocyte-concentratinghormone) antagonists, CCK (cholecystokinin) agonists, serotoninre-uptake inhibitors, serotonin and noradrenaline re-uptake inhibitors,mixed serotonin and noradrenergic compounds, 5HT (serotonin) agonists,bombesin agonists, galanin antagonists, growth hormone, growth hormonereleasing compounds, TRH (thyreotropin releasing hormone) agonists, UCP2 or 3 (uncoupling protein 2 or 3) modulators, leptin agonists, DAagonists (bromocriptin, doprexin), lipase/amylase inhibitors, RXR(retinoid X receptor) modulators, TR β agonists; histamine H3antagonists, Gastric Inhibitory Polypeptide agonists or antagonists (GIPanalogs), gastrin and gastrin analogs.

The treatment with a derivative according to this invention may also becombined with surgery—a surgery that influence the glucose levels and/orlipid homeostasis such as gastric banding or gastric bypass.

It should be understood that any suitable combination of the derivativesaccording to the invention with one or more of the above-mentionedcompounds and optionally one or more further pharmacologically activesubstances are considered to be within the scope of the presentinvention.

Method of Manufacturing

Depending on the sequence the analogues of this invention can beproduced by a method which comprises culturing a host cell containing aDNA sequence encoding the polypeptide and capable of expressing thepolypeptide in a suitable nutrient medium under conditions permittingthe expression of the peptide, after which the resulting peptide isrecovered from the culture.

The medium used to culture the cells may be any conventional mediumsuitable for growing the host cells, such as minimal or complex mediacontaining appropriate supplements. Suitable media are available fromcommercial suppliers or may be prepared according to published recipes(e.g. in catalogues of the American Type Culture Collection). Thepeptide produced by the cells may then be recovered from the culturemedium by conventional procedures including separating the host cellsfrom the medium by centrifugation or filtration, precipitating theproteinaceous components of the supernatant or filtrate by means of asalt, e.g. ammonium sulphate, purification by a variety ofchromatographic procedures, e.g. ion exchange chromatography, gelfiltration chromatography, affinity chromatography, or the like,dependent on the type of peptide in question.

The DNA sequence encoding the therapeutic polypeptide may suitably be ofgenomic or cDNA origin, for instance obtained by preparing a genomic orcDNA library and screening for DNA sequences coding for all or part ofthe polypeptide by hybridisation using synthetic oligonucleotide probesin accordance with standard techniques (see, for example, Sambrook, J,Fritsch, E F and Maniatis, T, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, New York, 1989). The DNA sequenceencoding the polypeptide may also be prepared synthetically byestablished standard methods, e.g. the phosphoamidite method describedby Beaucage and Caruthers, Tetrahedron Letters 22 (1981), 1859-1869, orthe method described by Matthes et al., EMBO Journal 3 (1984), 801-805.The DNA sequence may also be prepared by polymerase chain reaction usingspecific primers, for instance as described in U.S. Pat. No. 4,683,202or Saiki et al., Science 239 (1988), 487-491.

The DNA sequence may be inserted into any vector which may convenientlybe subjected to recombinant DNA procedures, and the choice of vectorwill often depend on the host cell into which it is to be introduced.Thus, the vector may be an autonomously replicating vector, i.e. avector which exists as an extrachromosomal entity, the replication ofwhich is independent of chromosomal replication, e.g. a plasmid.Alternatively, the vector may be one which, when introduced into a hostcell, is integrated into the host cell genome and replicated togetherwith the chromosome(s) into which it has been integrated.

The vector is preferably an expression vector in which the DNA sequenceencoding the peptide is operably linked to additional segments requiredfor transcription of the DNA, such as a promoter. The promoter may beany DNA sequence which shows transcriptional activity in the host cellof choice and may be derived from genes encoding proteins eitherhomologous or heterologous to the host cell. Examples of suitablepromoters for directing the transcription of the DNA encoding thepeptide of the invention in a variety of host cells are well-known inthe art, cf. for instance Sambrook et al., supra.

The DNA sequence encoding the peptide may also, if necessary, beoperably connected to a suitable terminator, polyadenylation signals,transcriptional enhancer sequences, and translational enhancersequences. The recombinant vector of the invention may further comprisea DNA sequence enabling the vector to replicate in the host cell inquestion.

The vector may also comprise a selectable marker, e.g. a gene theproduct of which complements a defect in the host cell or one whichconfers resistance to a drug, e.g. ampicillin, kanamycin, tetracyclin,chloramphenicol, neomycin, hygromycin or methotrexate.

To direct a parent peptide of the present invention into the secretorypathway of the host cells, a secretory signal sequence (also known as aleader sequence, prepro sequence or pre sequence) may be provided in therecombinant vector. The secretory signal sequence is joined to the DNAsequence encoding the peptide in the correct reading frame. Secretorysignal sequences are commonly positioned 5′ to the DNA sequence encodingthe peptide. The secretory signal sequence may be that normallyassociated with the peptide or may be from a gene encoding anothersecreted protein. The procedures used to ligate the DNA sequences codingfor the present peptide, the promoter and optionally the terminatorand/or secretory signal sequence, respectively, and to insert them intosuitable vectors containing the information necessary for replication,are well-known to persons skilled in the art (cf., for instance,Sambrook et al., supra).

The host cell into which the DNA sequence or the recombinant vector isintroduced may be any cell which is capable of producing the presentpeptide and includes bacteria, yeast, fungi and higher eukaryotic cells.Examples of suitable host cells well-known and used in the art are,without limitation, E. coli, Saccharomyces cerevisiae, or mammalian BHKor CHO cell lines.

Embodiments According to the Invention

-   1. A GLP-1 derivative which comprises a modified GLP-1 sequence 7-37    (SEQ ID No 1) having:    -   i) a total of 2, 3, 4, 5 or 6 amino acid substitutions compared        to the sequence 7-37 of SEQ ID No 1, including        -   a) a Glu residue at a position equivalent to position 22 of            SEQ ID No 1 and        -   b) an Arg residue at a position equivalent to position 26 of            SEQ ID No 1,    -   ii) optionally a C terminal extension of 1 amino acid residue,    -   iii) optionally the amino acid at a position equivalent to        position 37 of SEQ ID No 1 can be absent, and    -   iv) optionally a C-terminal amide group,    -   and which is derivatised with an albumin binding residue or        pegylated in a position selected from a position equivalent to        position 18, 20, 23, 30, 31, 34, 36 or 37 of SEQ ID No 1,        preferably at position 18, 23, 31, 34, 36 or 37 of SEQ ID No 1.-   2. The GLP-1 derivative according to embodiment 1 which comprises a    modified GLP-1 sequence 7-37 (SEQ ID NO 1) having:    -   i) a total of 2, 3, 4, 5 or 6 amino acid substitutions compared        to the sequence 7-37 of SEQ ID NO 1, including        -   a) a Glu residue at a position equivalent to position 22 of            SEQ ID No 1 and        -   b) an Arg residue at a position equivalent to position 26 of            SEQ ID No 1,    -   and which is derivatised with an albumin binding residue or        pegylated in a position selected from a position equivalent to        position 18, 20, 23, 30, 31, 34, 36 or 37 of SEQ ID No 1,        preferably at position 18, 23, 31, 34, 36 or 37 of SEQ ID No 1.-   3. The GLP-1 derivative according to embodiment 1, wherein the amino    acid at a position equivalent to position 37 of SEQ ID No 1 is    absent, and which is derivatised with an albumin binding residue or    pegylated in a position selected from at position equivalent to    position 18, 23, 31, 34 or 36 of SEQ ID NO 1,    -   and wherein the total length of the GLP-1 analogue is 30 amino        acids.-   4. The GLP-1 derivative according to embodiment 1 having a C    terminal extension of 1 amino acid residue in length and wherein the    total length of the GLP-1 analogue is 32 amino acids.-   5. The GLP-1 derivative according to any one of the embodiments 1-4    having a C-terminal amide group.-   6. The GLP-1 derivative according to any one of the embodiments 1-5    having the sequence of formula (I)

Formula (I) (SEQ ID No: 2)Xaa₇-Xaa₈-Xaa₉-Gly-Thr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Tyr-Xaa₂₀-Glu-Glu-Xaa₂₃-Xaa₂₄-Xaa₂₅-Arg-Xaa₂₇-Phe-Ile-Xaa₃₀-Xaa₃₁-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅- Xaa₃₆-Xaa₃₇-Xaa₃₈-R,

-   -   preferably having the sequence of formula (I′) which is        identical to formula (I) except for having Leu at position 20,    -   wherein    -   Xaa₇-Xaa₈ is L-histidine-Aib, desamino-histidine-alanine or        desamino-histidine-Aib    -   Xaa₉ is Glu or a Glu derivative such as alpha, alpha        dimethyl-Glu    -   Xaa₁₆ is Val or Leu;    -   Xaa₁₈ is Ser, Lys, Cys or Arg;    -   Xaa₂₀ is Leu or Lys    -   Xaa₂₃ is Gln, Glu, Lys, Cys or Arg;    -   Xaa₂₄ is Ala or Asn    -   Xaa₂₅ is Ala or Val;    -   Xaa₂₇ is Glu, Ala or Leu;    -   Xaa₃₀ is Ala, Glu, Lys or Arg, preferably Ala, Glu or Arg;    -   Xaa₃₁ is Trp, Cys or Lys;    -   Xaa₃₃ is Val, Cys or Lys;    -   Xaa₃₄ is Lys, Cys, Glu, Asn or Arg;    -   Xaa₃₅ is Gly or Aib;    -   Xaa₃₆ is Arg or Lys,    -   Xaa₃₇ is Gly, Aib, Cys, Lys or absent    -   Xaa₃₈ is Lys, Glu or absent;    -   R is amide or absent    -   provided that if Xaa₃₇ is absent, then Xaa₃₈ is also absent,    -   and which is derivatised with an albumin binding residue or        pegylated in a position selected from a position equivalent to        position 18, 20, 23, 30, 31, 34, 36 or 37 of SEQ ID No 1,        preferably at position 18, 23, 31, 34, 36 or 37 of SEQ ID No 1.

-   7. The GLP-1 derivative according to any one of the embodiments 1-5    having the sequence of formula (II)

Formula (II) (SEQ ID No: 3)Xaa₇-Xaa₈-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Xaa₁₈-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Arg-Glu-Phe-Ile-Xaa₃₀-Trp-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-R

-   -   wherein    -   Xaa₇ is L-histidine, D-histidine, desamino-histidine,        2-amino-histidine, β-hydroxy-histidine, homohistidine,        N^(α)-acetyl-histidine, α-fluoromethyl-histidine,        α-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or        4-pyridylalanine;    -   Xaa₈ is Ala, Gly, Val, Leu, Ile, Lys, Aib, (1-aminocyclopropyl)        carboxylic acid, (1-aminocyclobutyl) carboxylic acid,        (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl)        carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or        (1-aminocyclooctyl) carboxylic acid;    -   Xaa₁₈ is Ser, Lys or Arg;    -   Xaa₃₀ is Ala, Glu or Arg;    -   Xaa₃₃ is Val or Lys;    -   Xaa₃₄ is Lys, Glu or Arg;    -   Xaa₃₅ is Gly or Aib;    -   Xaa₃₆ is Arg or Lys,    -   Xaa₃₇ is Gly, Aib, Lys or absent,    -   Xaa₃₈ is Lys, Glu or absent,    -   R is amide or is absent    -   and which is derivatised with an albumin binding residue or        pegylated in a position selected from a position equivalent to        position 18, 20, 23, 30, 31, 34, 36 or 37 of SEQ ID No 1,        preferably at position 18, 23, 31, 34, 36 or 37 of SEQ ID No 1.

-   8. The GLP-1 derivative according to any one of the embodiments 1-7,    wherein at least one amino acid residue is derivatised with A-B-C-D-    -   wherein A- is selected from the group consisting of

-   -   wherein n is selected from the group consisting of 14, 15, 16        17, 18 and 19, p is selected from the group consisting of 10,        11, 12, 13 and 14, and d is selected from the group consisting        of 0, 1, 2, 3, 4 and 5,    -   —B— is selected from the group consisting of

-   -   wherein x is selected from the group consisting of 0, 1, 2, 3        and 4, and y is selected from the group consisting of 1, 2, 3,        4, 5, 6, 7, 8, 9, 10, 11 and 12,    -   —C— is selected from the group consisting of

-   -   wherein b and e are each independently selected from the group        consisting of 0, 1 and 2, and c and f are each independently        selected from the group consisting of 0, 1 and 2 with the        proviso that b is 1 or 2 when c is 0, or b is 0 when c is 1 or        2, and e is 1 or 2 when f is 0, or e is 0 when f is 1 or 2, and    -   -D- is attached to said amino acid residue and is a linker.

-   9. The GLP-1 derivative according to any one of embodiments 52-55,    wherein the derivatised amino acid residue is lysine.

-   10. A pharmaceutical composition comprising a derivative according    to any one of embodiments 1-9, and a pharmaceutically acceptable    excipient.

-   11. A derivative according to any one of the embodiments 1-9 for use    in the treatment or prevention of hyperglycemia, type 2 diabetes,    impaired glucose tolerance, type 1 diabetes, obesity, hypertension,    syndrome X, dyslipidemia, cognitive disorders, atherosclerosis,    myocardial infarction, coronary heart disease and other    cardiovascular disorders, stroke, inflammatory bowel syndrome,    dyspepsia and gastric ulcers.

The amino acid sequence of human GLP-1(7-37) is included in the SequenceListing as SEQ ID No: 1, and SEQ ID Nos: 2 and 3 are derivatives thereofaccording to the invention. In the Sequence Listing, the numberingstarts with amino acid residue no. 1. Accordingly, e.g., position 1 ofSEQ ID No 1 is equivalent to position 7 of GLP-1(7-37) (His), position16 of SEQ ID No 1 is equivalent to position 22 of GLP-1(7-37) (Gly), andposition 20 of SEQ ID No 1 is equivalent to position 26 of GLP-1(7-37)(Lys)- and vice versa for the other positions and the other sequences.

Accordingly, the invention also provides, in claim 1 of the priorityapplications, a GLP-1 derivative which comprises a modified GLP-1(7-37)sequence having:

i) a total of 2, 3, 4, 5 or 6 amino acid substitutions compared to thesequence of SEQ ID No 1, including

a) a Glu residue at a position equivalent to position 22 of GLP-1(7-37)(position 16 of SEQ ID No 1) and

b) an Arg residue at a position equivalent to position 26 of GLP-1(7-37)(position 20 of SEQ ID No 1),

ii) optionally a C terminal extension of 1 amino acid residue,

iii) optionally the amino acid at a position equivalent to position 37of GLP-1(7-37) (position 31 of SEQ ID No 1) can be absent, and

iv) optionally a C-terminal amide group,

and which is derivatised with an albumin binding residue or pegylated ina position selected from a position equivalent to position 18, 20, 23,30, 31, 34, 36 or 37 of GLP-1(7-37) (position 12, 14, 17, 24, 25, 28, 30or 31, respectively, of SEQ ID No 1);

The invention furthermore provides GLP-1 derivatives, methods and usesthereof, and pharmaceutical compositions with a content thereofcorresponding to any of the claims and particular embodiments accordingto the invention, in which corresponding position numbering amendmentshave been made as explained above, and shown above for the GLP-1derivative of claim 1 of the priority applications.

Additional Embodiments According to the Invention

-   1. A GLP-1 derivative which comprises a modified GLP-1 sequence 7-37    (SEQ ID No 1) having:    -   i) a total of 2, 3, 4, 5 or 6 amino acid substitutions compared        to the sequence 7-37 of SEQ ID No 1, including        -   a) a Glu residue at a position equivalent to position 22 of            SEQ ID No 1 and        -   b) an Arg residue at a position equivalent to position 26 of            SEQ ID No 1,    -   ii) optionally a C terminal extension of 1 amino acid residue,    -   iii) optionally the amino acid at a position equivalent to        position 37 of SEQ ID No 1 can be absent, and    -   iv) optionally a C-terminal amide group,    -   and which is derivatised with an albumin binding residue or        pegylated in a position selected from a position equivalent to        position 18, 20, 23, 30, 31, 34, 36 or 37 of SEQ ID No 1,        preferably 18, 23, 31, 34, 36 or 37 of SEQ ID No 1.

In embodiments of the invention a maximum of 6 amino acids have beenmodified. In embodiments of the invention a maximum of 5 amino acidshave been modified. In embodiments of the invention a maximum of 4 aminoacids have been modified. In embodiments of the invention a maximum of 3amino acids have been modified. In embodiments of the invention amaximum of 2 amino acids have been modified. In embodiments of theinvention 1 amino acid has been modified. In embodiments of theinvention one amino acid has been added in the C-terminal. Inembodiments of the invention one amino acid has been deleted in theC-terminal. In embodiments of the invention, there is a C-terminal amidegroup.

-   2. The GLP-1 derivative according to embodiment 1 which comprises a    modified GLP-1 sequence 7-37 (SEQ ID NO 1) having:    -   i) a total of 2, 3, 4, 5 or 6 amino acid substitutions compared        to the sequence 7-37 of SEQ ID NO 1, including        -   a) a Glu residue at a position equivalent to position 22 of            SEQ ID No 1 and        -   b) an Arg residue at a position equivalent to position 26 of            SEQ ID No 1,    -   and which is derivatised with an albumin binding residue or        pegylated in a position selected from a position equivalent to        position 18, 20, 23, 30, 31, 34, 36 or 37 of SEQ ID No 1,        preferably 18, 23, 31, 34, 36 or 37 of SEQ ID No 1.-   3. The GLP-1 derivative according to embodiment 1, wherein the amino    acid at a position equivalent to position 37 of SEQ ID No 1 is    absent, and which is derivatised with an albumin binding residue or    pegylated in a position selected from at position equivalent to    position 18, 23, 31, 34 or 36 of SEQ ID NO 1,    -   and wherein the total length of the GLP-1 analogue is 30 amino        acids.-   4. The GLP-1 derivative according to embodiment 1 having a C    terminal extension of 1 amino acid residue in length and wherein the    total length of the GLP-1 analogue is 32 amino acids.-   5. The GLP-1 derivative according to any one of the embodiments 1-4    having a C-terminal amide group.-   6. The GLP-1 derivative according to any one of the embodiments 1-5    having 3 amino acid substitutions compared to the sequence 7-37 of    SEQ ID NO 1 including the substitutions in position 22 and 26.-   7. The GLP-1 derivative according to any one of the embodiments 1-6,    which has a substitution selected at a position from the group of    positions 7, 8, 18, 20, 23, 24, 25, 27, 30, 31, 33, 34 and 37    compared to the sequence 7-37 of SEQ ID NO 1.-   8. The GLP-1 derivative according to embodiment 7, which has a    substitution selected from the group consisting of desaminoHis7,    Aib8, Lys18, Cys18, Lys20, Cys20, Lys23, Cys23, Asn24, Val25, Ala27,    Leu27, Glu30, Lys31, Cys31, Lys33, Cys33, Lys34, Cys34, Asn34, Cys37    and Lys37.-   9. The GLP-1 derivative according to any one of the embodiments 7-8,    which has a substitution selected from the group consisting of    desaminoHis7, Aib8, Lys18, Lys20, Lys23, Glu30, Lys31, Lys33, Lys 34    and Lys 37.-   10. The GLP-1 derivative according to any one of the embodiments    7-9, which has a substitution selected from the group consisting of    desaminoHis7 and Aib8.-   11. The GLP-1 derivative according to any one of the embodiments 1-5    having 4 amino acid substitutions compared to the sequence 7-37 of    SEQ ID NO 1 including the substitutions in position 22 and 26.-   12. The GLP-1 derivative according to any one of the embodiments 1-5    and 11, which has two substitutions at a position selected from the    group of positions 7, 8, 18, 20, 23, 24, 25, 27, 30, 31, 33, 34 and    37 compared to the sequence 7-37 of SEQ ID NO 1.-   13. The GLP-1 derivative according to any one of the embodiments    11-12, which has two substitutions selected from the group    consisting of desaminoHis7, Aib8, Lys18, Cys18, Lys20, Cys20, Lys23,    Cys23, Asn24, Val25, Ala27, Leu27, Glu30, Lys31, Cys31, Lys33,    Cys33, Lys34, Cys34, Asn34, Cys37 and Lys37.-   14. The GLP-1 derivative according to any one of the embodiments    11-13 having an amino acid substitution selected from the group    consisting of desaminoHis7 and Aib8 and an amino acid substitution    selected from the group consisting Lys18, Lys20, Lys23, Glu30,    Lys31, Lys33, Lys 34 and Lys 37.-   15. The GLP-1 derivative according to any one of the embodiments    11-14 having an amino acid substitution selected from the group    consisting desaminoHis7 and Aib8, and an amino acid substitution    selected from the group consisting Lys18, Lys20, Lys23, Glu30,    Lys31, Lys33, Lys 34 and Lys 37.-   16. The GLP-1 derivative according to any one of the embodiments 1-5    having 5 amino acid substitutions compared to the sequence 7-37 of    SEQ ID NO 1 including the substitutions in position 22 and 26.-   17. The GLP-1 derivative according to any one of the embodiments 1-5    and 16, which has three amino acid substitutions at a position    selected from the group of positions 7, 8, 18, 20, 23, 24, 25, 27,    30, 31, 33, 34 and 37 compared to the sequence 7-37 of SEQ ID NO 1.-   18. The GLP-1 derivative according to any one of the embodiments    16-17, which has three amino acid substitutions selected from the    group of desaminoHis7, Aib8, Lys18, Cys18, Lys20, Cys20, Lys23,    Cys23, Asn24, Val25, Ala27, Leu27, Glu30, Lys31, Cys31, Lys33,    Cys33, Lys34, Cys34, Asn34, Cys37 and Lys37.-   19. The GLP-1 derivative according to any one of the embodiments    16-18 having an amino acid substitution selected from the group    consisting of desaminoHis7 and Aib8, and two amino acid    substitutions selected from the group consisting of Lys18, Lys20,    Lys23, Glu30, Lys31, Lys33, Lys 34 and Lys 37.-   20. The GLP-1 derivative according to any one of the embodiments    16-19 having an amino acid substitution selected from the group    consisting of desaminoHis7 and Aib8, and two amino acid    substitutions selected from the group consisting of Lys18, Lys20,    Lys23, Glu30, Lys31, Lys33, Lys 34 and Lys 37.-   21. The GLP-1 derivative according to any one of the embodiments 1-5    having 6 amino acid substitutions compared to the sequence 7-37 of    SEQ ID NO 1 including the substitutions in position 22 and 26.-   22. The GLP-1 derivative according to any one of the embodiments 1-5    and 21, which has four amino acid substitutions at a position    selected from the group of positions 7, 8, 18, 20, 23, 24, 25, 27,    30, 31, 33, 34 and 37.-   23. The GLP-1 derivative according to any one of the embodiments    21-22, which has four amino acid substitutions selected from the    group consisting of desaminoHis7, Aib8, Lys18, Cys18, Lys20, Cys20,    Lys23, Cys23, Asn24, Val25, Ala27, Leu27, Glu30, Lys31, Cys31,    Lys33, Cys33, Lys34, Cys34, Asn34, Cys37 and Lys37.-   24. The GLP-1 derivative according to any one of the embodiments    21-23 having an amino acid substitution selected from the group    consisting of desaminoHis7 and Aib8 and three amino acid    substitutions selected from the group consisting of Lys18, Lys20,    Lys23, Glu30, Lys31, Lys33, Lys34 and Lys37.-   25. The GLP-1 derivative according to any one of the embodiments    21-24 having an amino acid substitution selected from the group    consisting of desaminoHis7 and Aib8 and three amino acid    substitutions selected from the group consisting of Lys18, Lys20,    Lys23, Glu30, Lys31, Lys33, Lys34 and Lys37.-   26. The GLP-1 derivative according to any one of the embodiments    1-25, which has been pegylated or derivatised with an albumin    binding residue in position 18.-   27. The GLP-1 derivative according to any one of the embodiments    1-26, which has been pegylated or derivatised with an albumin    binding residue in position 23.-   28. The GLP-1 derivative according to any one of the embodiments    1-26, which has been pegylated or derivatised with an albumin    binding residue in position 31.-   29. The GLP-1 derivative according to any one of the embodiments    1-26, which has been pegylated or derivatised with an albumin    binding residue in position 34.-   30. The GLP-1 derivative according to any one of the embodiments    1-26, which has been pegylated or derivatised with an albumin    binding residue in position 36.-   31. The GLP-1 derivative according to any one of the embodiments    1-26, which has been pegylated or derivatised with an albumin    binding residue in position 37.-   32. The GLP-1 derivative according to any one of the embodiments    1-31, which has been pegylated.-   33. The GLP-1 derivative according to any one of the embodiments    1-32, wherein the amino acid at a position equivalent to position 37    of SEQ ID No 1 has been substituted with Lys, and which Lys residue    is pegylated or derivatised with an albumin binding residue.-   34. The GLP-1 derivative according to any one of the embodiments    1-33, which has been derivatised with an albumin binding residue.-   35. The GLP-1 derivative according to any one of the embodiments    1-34 having the sequence of formula (I)

Xaa₇-Xaa₈-Xaa₉-Gly-Thr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Tyr-Xaa₂₀-Glu-Glu-Xaa₂₃-Xaa₂₄-Xaa₂₅-Arg-Xaa₂₇-Phe-Ile-Xaa₃₀-Xaa₃₁-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅- Xaa₃₆-Xaa₃₇-Xaa₃₈-R

-   -   Formula (I) (SEQ ID No: 2), preferably Formula (I′) which is        identical to Formula (I) except for having a Leu at position 20,    -   Wherein    -   Xaa₇-Xaa₈ is L-histidine-Aib, desamino-histidine-alanine or        desamino-histidine-Aib    -   Xaa₉ is Glu or a Glu derivative such as alpha, alpha        dimethyl-Glu    -   Xaa₁₆ is Val or Leu;    -   Xaa₁₈ is Ser, Lys, Cys or Arg;    -   Xaa₂₀ is Leu or Lys    -   Xaa₂₃ is Gln, Glu, Lys, Cys or Arg;    -   Xaa₂₄ is Ala or Asn    -   Xaa₂₅ is Ala or Val;    -   Xaa₂₇ is Glu, Ala or Leu;    -   Xaa₃₀ is Ala, Glu, Lys or Arg, preferably Ala, Glu or Arg;    -   Xaa₃₁ is Trp, Cys or Lys;    -   Xaa₃₃ is Val, Cys or Lys;    -   Xaa₃₄ is Lys, Cys, Glu, Asn or Arg;    -   Xaa₃₅ is Gly or Aib;    -   Xaa₃₆ is Arg or Lys,    -   Xaa₃₇ is Gly, Aib, Cys, Lys or absent    -   Xaa₃₈ is Lys, Glu or absent;    -   R is amide or absent    -   provided that if Xaa₃₇ is absent, then Xaa₃₈ is also absent,    -   and which is derivatised with an albumin binding residue or        pegylated in a position selected from a position equivalent to        position 18, 20, 23, 30, 31, 34, 36 or 37 of SEQ ID No 1,        preferably 18, 23, 31, 34, 36 or 37 of SEQ ID No 1.

-   36. The GLP-1 derivative according to any one of the embodiments    1-35 having the sequence of formula (II)

Formula (II) (SEQ ID No: 3)Xaa₇-Xaa₈-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Xaa₁₈-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Arg-Glu-Phe-Ile-Xaa₃₀-Trp-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-R

-   -   wherein    -   Xaa₇ is L-histidine, D-histidine, desamino-histidine,        2-amino-histidine, β-hydroxy-histidine, homohistidine,        N^(α)-acetyl-histidine, α-fluoromethyl-histidine,        α-methyl-histidine, 3-pyridylalanine, 2-pyridylalanine or        4-pyridylalanine;    -   Xaa₈ is Ala, Gly, Val, Leu, Ile, Lys, Aib, (1-aminocyclopropyl)        carboxylic acid, (1-aminocyclobutyl) carboxylic acid,        (1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl)        carboxylic acid, (1-aminocycloheptyl) carboxylic acid, or        (1-aminocyclooctyl) carboxylic acid;    -   Xaa₁₈ is Ser, Lys or Arg;    -   Xaa₃₀ is Ala, Glu or Arg;    -   Xaa₃₃ is Val or Lys;    -   Xaa₃₄ is Lys, Glu or Arg;    -   Xaa₃₅ is Gly or Aib;    -   Xaa₃₆ is Arg or Lys,    -   Xaa₃₇ is Gly, Aib, Lys or absent    -   Xaa₃₈ is Lys, Glu or absent;    -   R is amide or is absent    -   and which is derivatised with an albumin binding residue or        pegylated in a position selected from a position equivalent to        position 18, 20, 23, 30, 31, 34, 36 or 37 of SEQ ID No 1,        preferably at position 18, 23, 31, 34, 36 or 37 of SEQ ID No 1.

-   37. The GLP-1 derivative according to any one of the embodiments    35-36, wherein Xaa₃₈ is absent.

-   38. The GLP-1 derivative according to any one of the embodiments    35-37, wherein Xaa₃₇ and Xaa₃₈ are both absent.

-   39. The GLP-1 derivative according to any one of the embodiments    35-38, wherein Xaa₃₇ and Xaa₃₈ are both absent, and Xaa₃₆ is    Xaa₃₆-amide.

-   40. The GLP-1 derivative according to any one of the embodiments    35-39, wherein 3 amino acids are substituted compared to the    sequence 7-37 of SEQ ID NO 1 and where Xaa₇ is desamino-histidine.

-   41. The GLP-1 derivative according to any one of the embodiments    35-39, wherein 4 amino acids are substituted compared to the    sequence 7-37 of SEQ ID NO 1 and where Xaa₇ is desamino-histidine.

-   42. The GLP-1 derivative according to any one of the embodiments    35-39, wherein 5 amino acids are substituted compared to the    sequence 7-37 of SEQ ID NO 1 and where Xaa₇ is desamino-histidine.

-   43. The GLP-1 derivative according to any one of the embodiments    35-39, wherein 6 amino acids are substituted compared to the    sequence 7-37 of SEQ ID NO 1 and where Xaa₇ is desamino-histidine.

-   44. The GLP-1 derivative according to any one of the embodiments    35-39, wherein 3 amino acids are substituted compared to the    sequence 7-37 of SEQ ID NO 1 and where Xaa₈ is Aib.

-   45. The GLP-1 derivative according to any one of the embodiments    35-39, wherein 4 amino acids are substituted compared to the    sequence 7-37 of SEQ ID NO 1 and where Xaa₈ is Aib.

-   46. The GLP-1 derivative according to any one of the embodiments    35-39, wherein 5 amino acids are substituted compared to the    sequence 7-37 of SEQ ID NO 1 and where Xaa₈ is Aib.

-   47. The GLP-1 derivative according to any one of the embodiments    35-39, wherein 6 amino acids are substituted compared to the    sequence 7-37 of SEQ ID NO 1 and where Xaa₈ is Aib.

-   48. The GLP-1 derivative according to any one of the embodiments    35-47, wherein Xaa₇ is desamino-histidine.

-   49. The GLP-1 derivative according to any one of the embodiments    1-48, which comprises a hydrophilic spacer between the modified    GLP-1 sequence and one or more albumin binding residue(s).

-   50. The GLP-1 derivative according to embodiment 49, wherein the    hydrophilic spacer is an unbranched oligo ethylene glycol moiety    with appropriate functional groups at both terminals that forms a    bridge between an amino group of the modified GLP-1 sequence and a    functional group of the albumin binding residue.

-   51. The GLP-1 derivative according to any one of the embodiments    1-59, which has been derivatised with an albumin binding residue.

-   52. The GLP-1 derivative according to any one of the embodiments    1-51, wherein at least one amino acid residue is derivatised with    A-B-C-D-    -   wherein A- is selected from the group consisting of

and tetrazole without sulphonamide,

-   -   wherein n is selected from the group consisting of 14, 15, 16        17, 18 and 19, p is selected from the group consisting of 10,        11, 12, 13 and 14, and d is selected from the group consisting        of 0, 1, 2, 3, 4 and 5,    -   —B— is selected from the group consisting of

-   -   wherein x is selected from the group consisting of 0, 1, 2, 3        and 4, and y is selected from the group consisting of 1, 2, 3,        4, 5, 6, 7, 8, 9, 10, 11 and 12,    -   —C— is selected from the group consisting of

-   -   wherein b and e are each independently selected from the group        consisting of 0, 1 and 2, and c and f are each independently        selected from the group consisting of 0, 1 and 2 with the        proviso that b is 1 or 2 when c is 0, or b is 0 when c is 1 or        2, and e is 1 or 2 when f is 0, or e is 0 when f is 1 or 2, and    -   -D- is attached to said amino acid residue and is a linker.

-   53. The GLP-1 derivative according to embodiment 52, wherein one    amino acid residue is derivatised with A-B-C-D-.

-   54. The GLP-1 derivative according to any one of embodiments 52-53,    wherein the derivatised amino acid residue comprises an amino group.

-   55. The GLP-1 derivative according to any one of embodiments 52-54,    wherein the derivatised amino acid residue comprises a primary amino    group in a side chain.

-   56. The GLP-1 derivative according to any one of embodiments 52-55,    wherein the derivatised amino acid residue is lysine.

-   57. The GLP-1 derivative according to any one of embodiments 52-56,    wherein only one amino acid residue is derivatised.

-   58. The GLP-1 derivative according to any one of embodiments 52-57,    wherein A- is

-   59. The GLP-1 derivative according to any of the embodiments 52-58,    wherein n is selected from the group consisting of 15 and 17, and    more is preferred 17.-   60. The GLP-1 derivative according to any one of the embodiments    52-57, wherein A- is

-   61. The GLP-1 derivative according to any of the embodiments 52-57    and 60, wherein p is selected from the group consisting of 12, 13,    and 14 and more preferred is 13.-   62. The GLP-1 derivative according to any of the embodiments 52-57    and 60-61, wherein d is selected from the group consisting of 0, 1,    2, 3 and 4, more preferred 0, 1 and 2 and most preferred 1.-   63. The GLP-1 derivative according to any of the embodiments 52-57    and 52-62, wherein d is selected from the group consisting of 0, 1    and 2 and p is selected from the group consisting of 12, 13 or 14,    more preferred d is selected from the group consisting of 1 and 2    and p is selected from the group consisting of 13 and 14, and most    preferred d is 1 and p is 13.-   64. The GLP-1 derivative according to any of the embodiments 52-63,    wherein —B— is

-   65. The GLP-1 derivative according to any of the embodiments 52-63,    wherein —B— is

-   66. The GLP-1 derivative according to any of the embodiments 52-63,    wherein —B— is

-   67. The GLP-1 derivative according to any of the embodiments 52-63,    wherein —B— is

-   68. The GLP-1 derivative according to embodiment 67, wherein x is    selected from the group consisting of 0, 1 and 2, more preferred x    is selected from the group consisting of 0 and 1 and most preferred    x is 0, or 1, preferably 0.-   69. The GLP-1 derivative according to any of the embodiments 52-63,    wherein —B— is

-   70. The GLP-1 derivative according to embodiment 69, wherein y is    selected from the group consisting of 2, 3, 4, 5, 6, 7, 8, 9 and 10    and more preferred y is selected from the group consisting of 2, 3,    4, 5, 6, 7, and 8.-   71. The GLP-1 derivative according to any of the embodiments 52-70,    wherein —C— is

-   72. The GLP-1 derivative according to embodiment 71, wherein c is    selected from the group consisting of 0 and 1 and b is selected from    the group consisting of 1 and 2, more preferred b is 1, or 2,    preferably 2; and c is 0.-   73. The GLP-1 derivative according to any of the embodiments 52-70,    wherein —C— is

-   74. The GLP-1 derivative according to embodiment 73, wherein f is    selected from the group consisting of 0 and 1 and e is selected from    the group consisting of 1 and 2, more preferred e is 1 and f is 0.-   75. The GLP-1 derivative according to any of the embodiments 52-70,    wherein —C— is

-   76. The GLP-1 derivative according to any of the embodiments 52-70,    wherein D is selected from the group consisting of

-   -   and wherein k is selected from the group consisting of 0, 1, 2,        3, 4, 5, 11 and 27,    -   and m is selected from the group consisting of 0, 1, 2, 3, 4, 5        and 6.

-   77. The GLP-1 derivative according to any of the embodiments 52-76,    wherein -D- is

-   78. The GLP-1 derivative according to embodiment 77, wherein k is    selected from the group consisting of 1, 2, 3, 11 and 27 and more    preferred k is 1.-   79. The GLP-1 derivative according to any of the embodiments 77-78,    wherein m is selected from the group consisting of 0, 1, 2, 3, and 4    and more preferred m is selected from the group consisting of 0, 1    and 2.-   80. The GLP-1 derivative according to any of the embodiments 52-76,    wherein -D- is

-   81. The GLP-1 derivative according to embodiment 80, wherein m is    selected from the group consisting of 0, 1, 2, 3, and 4 and more    preferred m is selected from the group consisting of 0, 1 and 2.-   82. The GLP-1 derivative according to any of the embodiments 52-76,    wherein -D- is

-   83. The GLP-1 derivative according to embodiment 82, wherein m is    selected from the group consisting of 0, 1, 2, 3, and 4 and more    preferred m is selected from the group consisting of 0, 1 and 2.-   84. The GLP-1 derivative according to any of the embodiments 52-76,    wherein -D- is

-   85. The GLP-1 derivative according to embodiment 84, wherein m is    selected from the group consisting of 0, 1, 2, 3, and 4 and more    preferred m is selected from the group consisting of 0, 1 and 2.-   86. The GLP-1 derivative according to any of the embodiments 52-76,    wherein -D- is

-   87. The GLP-1 derivative according to embodiment 86, wherein m is    selected from the group consisting of 0, 1, 2, 3, and 4 and more    preferred m is selected from the group consisting of 0, 1 and 2.-   88. The GLP-1 derivative according to any of the embodiments 52-76,    wherein -D- is

-   89. The GLP-1 derivative according to embodiment 88, wherein m is    selected from the group consisting of 0, 1, 2, 3, and 4 and more    preferred m is selected from the group consisting of 0, 1 and 2.-   90. The GLP-1 derivative according to any of the embodiments 52-76,    wherein A-B-C-D- is selected and combined from

-   91. The GLP-1 derivative according to any of the embodiments 52-76,    wherein A-B-C-D- is selected and combined from

-   92. The GLP-1 derivative according to any of the embodiments 52-76,    wherein A-B-C-D- is selected from the group consisting of

-   93. The derivative according to any of the above embodiments, which    is selected from the group consisting of-   N-epsilon37{2-[2-(2-{2-[2-((R)-3-carboxy-3-{[1-(19-carboxynonadecanoyl)piperidine-4-carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl    [desaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1(7-37)amide,-   N-epsilon37{2-[2-(2-{2-[2-((S)-3-carboxy-3-{[1-(19-carboxynonadecanoyl)piperidine-4-carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl    [Aib8,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide,-   N-epsilon37-[2-(2-[2-(2-[2-(2-((R)-3-[1-(17-Carboxyheptadecanoyl)piperidin-4-ylcarbonylamino]3-carboxypropionylamino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][,Desamin    oHis7, Glu22 Arg26, Arg 34, Phe(m-CF₃)28]GLP-1-(7-37)amide,-   N-epsilon30{2-[2-(2-{2-[2-((S)-3-carboxy-3-{[1-(19-carboxynonadecanoyl)piperidine-4-carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl    [Aib8,Glu22,Arg26,Lys30]GLP-1-(7-37),-   N-epsilon31{2-[2-(2-{2-[2-((S)-3-carboxy-3-{[1-(19-carboxynonadecanoyl)piperidine-4-carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl    [Aib8, Glu22, Arg26,Lys 31]GLP-1-(7-37),-   N-epsilon31-(2-{2-[2-(2-{2-[2-((S)-3-Carboxy-3-{[1-(19-carboxy-nonadecanoyl)piperidine-4-carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl)[Aib8,Glu22,Arg26,Lys31,Arg34]GLP-1-(7-37),-   N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxy-nonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide,-   N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxy-nonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide,-   N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxy-nonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37),-   N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxy-nonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg26,Glu30,Arg34,Lys37    ]GLP-1-(7-37),-   N-epsilon20-[2-(2-{2-[(S)-4-Carboxy-4-((S)-4-carboxy-4-{12-[4-(16-(1H-tetrazol-5-yl)hexadecanoylsulfamoyl)butyrylamino]dodecanoylamino}butyrylamino)butyrylamino]ethoxy}ethoxy)acetyl][Aib8,Lys20,Glu22,Arg26,Glu30,Pro37]GLP-1-(7-37)amide,-   N-epsilon37-[2-(2-{2-[(S)-4-Carboxy-4-((S)-4-carboxy-4-{12-[4-(16-(1H-tetrazol-5-yl)hexadecanoylsulfamoyl)butyrylamino]dodecanoylamino}butyrylamino)butyrylamino]ethoxy}ethoxy)acetyl][Aib8,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide,-   N-epsilon37-[2-(2-{2-[(S)-4-Carboxy-4-((S)-4-carboxy-4-{12-[4-(16-(1H-tetrazol-5-yl)hexadecanoylsulfamoyl)butyrylamino]dodecanoylamino}butyrylamino)butyrylamino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide,-   [Aib8,Glu22,Arg26,Glu30,Pro37]GLP-1-(7-37)Lys    [2-(2-{2-[4-Carboxy-4-(4-carboxy-4-{4-[4-(16-1H-tetrazol-5-yl-hexadecanoylsulfamoyl)butyrylamino]butyrylamino}butyrylamino)butyrylamino]ethoxy}ethoxy)acetyl],-   N-epsilon37    (Polyethyleneglycol2000)[DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1    (7-37)amide,-   N-epsilon37    (3-((2-(2-(2-(2-(2-Hexadecyloxyethoxy)ethoxy)ethoxy)ethoxy)ethoxy))propionyl)[DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1(7-37)-amide,-   N-epsilon37-{2-(2-(2-(2-[2-(2-(4-(hexadecanoylamino)-4-carboxybutyrylamino)ethoxy)ethoxy]acetyl)ethoxy)ethoxy)acetyl)}-[desaminoHis7,Glu22,Arg26,    Glu30,Arg34,Lys37] (GLP-1-(7-37)amide,-   N-epsilon37-{2-(2-(2-(2-[2-(2-(4-(hexadecanoylamino)-4-carboxybutyrylamino)ethoxy)ethoxy]acetyl)ethoxy)ethoxy)acetyl)}-[desaminoHis7,Glu22,    Arg26,Arg34,Lys 37] (GLP-1-(7-37)amide,-   N-epsilon37-(2-(2-(2-(2-(2-(2-(2-(2-(2-(Octadecanoylamino)ethoxy)ethoxy)acetylamino)ethoxy)ethoxy)acetylamino)ethoxy)ethoxy)acetyl)[desaminoHis7,Glu22,Arg26,Arg34,Lys37]    GLP-1 (7-37)amide,-   N-epsilon36-(2-(2-(2-((2-[2-(2-(17-carboxyheptadecanoylamino)ethoxy)ethoxy]acetylamino)ethoxy)ethoxy)acetyl)    [Aib8,Glu22,Arg26,Glu30,Lys36] GLP-1-(7-37)Glu-amide,-   N-epsilon37-[4-(16-(1H-Tetrazol-5-yl)hexadecanoylsulfamoyl)butyryl][DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide,-   N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(19-carboxynonadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37),    and-   N-epsilon31-[2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Glu22,Arg26,Lys31]GLP-1-(7-37).-   94. A method for increasing the time of action in a patient of a    GLP-1 analogue, characterised in that a modified GLP-1 sequence 7-37    (SEQ ID No 1) is derivatised or pegylated as disclosed in any of the    preceding embodiments.-   95. A method for increasing the time of action in a patient of a    GLP-1 derivative to more than about 40 hours, characterised in that    a modified GLP-1 sequence 7-37 (SEQ ID No 1) is derivatised or    pegylated as disclosed in any of the preceding embodiments.-   96. A pharmaceutical composition comprising a derivative according    to any one of embodiments 1-93, and a pharmaceutically acceptable    excipient.-   97. The pharmaceutical composition according to embodiment 96, which    is suited for parenteral administration.-   98. Use of a derivative according to any one of the embodiments 1-93    for the preparation of a medicament.-   99. Use of a derivative according to any one of the embodiments 1-93    for the preparation of a medicament for the treatment or prevention    of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type    1 diabetes, obesity, hypertension, syndrome X, dyslipidemia,    cognitive disorders, atherosclerosis, myocardial infarction,    coronary heart disease and other cardiovascular disorders, stroke,    inflammatory bowel syndrome, dyspepsia and gastric ulcers.-   100. Use of a derivative according to any one of the embodiments    1-93 for the preparation of a medicament for delaying or preventing    disease progression in type 2 diabetes.-   101. Use of a derivative according to any one of the embodiments    1-93 for the preparation of a medicament for decreasing food intake,    decreasing β-cell apoptosis, increasing β-cell function and β-cell    mass, and/or for restoring glucose sensitivity to β-cells.-   102. A derivative according to any one of the embodiments 1-93 for    use in the treatment or prevention of hyperglycemia, type 2    diabetes, impaired glucose tolerance, type 1 diabetes, obesity,    hypertension, syndrome X, dyslipidemia, cognitive disorders,    atherosclerosis, myocardial infarction, coronary heart disease and    other cardiovascular disorders, stroke, inflammatory bowel syndrome,    dyspepsia and gastric ulcers.

Further Particular Embodiments According to the Invention

-   1. A GLP-1 derivative which comprises a modified GLP-1(7-37)    sequence having:    -   i) a total of 2-12 amino acid modifications as compared to        GLP-1(7-37) (SEQ ID No: 1), including        -   a) a Glu residue at a position equivalent to position 22 of            GLP-1(7-37), and        -   b) an Arg residue at a position equivalent to position 26 of            GLP-1(7-37);            and    -   ii) which is derivatised with an albumin binding residue or        pegylated in a position selected from a position equivalent to        position 18, 20, 23, 30, 31, 34, 36, 37, or 39 of GLP-1(7-37).-   2. The GLP-1 derivative according to embodiment 1 which comprises 2,    3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid modifications;    preferably 4-12, such as 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acid    modifications; or such as 4, 5, 6, 7, 8, 9, or 12 amino acid    modifications; more preferably 5-11 amino acid modifications; even    more preferably 6-10 amino acid modifications; most preferably 7-9    amino acid modifications.-   3. The GLP-1 derivative according to any one of embodiments 1-2    which comprises 2-8, preferably 2, 3, 4, 5, 6, 7, or 8, amino acid    substitutions as compared to GLP-1(7-37) (SEQ ID No: 1).-   4. The GLP-1 derivative according to embodiment 3 which comprises    3-8, preferably 4-8, more preferably 5-8, even more preferably 6-8,    and most preferably 7-8 amino acid substitutions.-   5. The GLP-1 derivative according to embodiment 3 which comprises    i)4, ii)5, iii)6, iv)7, or v) 8 amino acid substitutions.-   6. The GLP-1 derivative according to any one of embodiments 3-5    which is substituted at one or more of positions 7, 8, 20, 25, 28,    30, 31, 34, 35, 36, or 37, preferably at position 7 and/or 8.-   7. The GLP-1 derivative according to any one of embodiments 1-6    which comprises a non-natural amino acid at position 7 or 8.-   8. The GLP-1 derivative according to embodiment 7, which comprises    7-Desamino-histidine or 8-Aib.-   9. The GLP-1 derivative according to any one of embodiments 1-7,    which comprises one or more of 7-Desamino-histidine, 8-Aib, 20K,    25V, 28F(m-CF₃), 30E, 30K, 31K, 34-Dap, 34R, 35-Aib, 35K, 35R, 36K,    37K, 37K-epsilon, 37P, and/or 37R.-   10. The GLP-1 derivative according to any one of embodiments 1-9    which comprises 1-3, preferably 1, 2, or 3, amino acid deletions as    compared to GLP-1(7-37) (SEQ ID No: 1).-   11. The GLP-1 derivative according to embodiment 10 wherein the    deletion is C-terminal.-   12. The GLP-1 derivative according to embodiment 11 which is a    GLP-1(7-36), a GLP-1(7-35), or a GLP-1(7-34) derivative.-   13. The GLP-1 derivative according to any one of embodiments 1-9    which comprises 1-4, preferably 1, 2, 3, or 4, amino acid additions    as compared to GLP-1(7-37) (SEQ ID No: 1).-   14. The GLP-1 derivative according to embodiment 13 wherein the    addition is C-terminal.-   15. The GLP-1 derivative according to embodiment 14 which is a    GLP-1(7-38), a GLP-1(7-39), a GLP-1(7-40), or a GLP-1(7-41)    derivative.-   16. The GLP-1 derivative according to any one of the embodiments    1-15 which has (i) a C-terminal amide group; or (ii) a C-terminal    carboxylic acid group, preferably a free carboxylic acid group    (—COOH), or a salt thereof.-   17. A GLP-1 derivative, preferably according to any one of the    embodiments 1-16, having the sequence of formula (I)

Formula (I) (SEQ ID No: 2)Xaa₇-Xaa₈-Xaa₉-Gly-Thr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Tyr-Xaa₂₀-Glu-Glu-Xaa₂₃-Xaa₂₄-Xaa₂₅-Arg-Xaa₂₇-Xaa₂₈-Ile-Xaa₃₀-Xaa₃₁-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-Xaa₄₀-Xaa₄₁-Rwherein(Xaa₇-Xaa₈) is (L-histidine-Aib), (desamino-histidine-alanine), or(desamino-histidine-Aib);Xaa₉ is Glu, or a Glu derivative such as alpha, alpha dimethyl-Glu;Xaa₁₆ is Val, or Leu;Xaa₁₈ is Ser, Lys, Cys, or Arg;Xaa₂₀ is Leu, or Lys;Xaa₂₃ is Gln, Glu, Lys, Cys, or Arg;Xaa₂₄ is Ala, or Asn;Xaa₂₅ is Ala, or Val;Xaa₂₇ is Glu, Ala, or Leu;Xaa₂₈ is Phe, or a Phe derivative such as m-CF₃-Phe;Xaa₃₀ is Ala, Glu, Lys, or Arg;Xaa₃₁ is Trp, Cys, or Lys;Xaa₃₃ is Val, Cys, or Lys;Xaa₃₄ is Lys, Cys, Glu, Asn, Dap, or Arg;Xaa₃₅ is Gly, Arg, Lys, Aib, or absent;Xaa₃₆ is Arg, Lys, or absent;Xaa₃₇ is Gly, Aib, Cys, Lys, epsilon-amino-Lys, Pro, Arg, or absent;Xaa₃₈ is Lys, Glu, Arg, or absent;Xaa₃₉ is Lys, Arg, or absent;Xaa₄₀ is Arg, or absent;Xaa₄₁ is Arg, or absent; andR is amide, or absent;provided that if Xaa₃₇, Xaa₃₈, Xaa₃₉, or Xaa₄₀ is absent, then eachamino acid residue downstream is also absent;and which is derivatised with an albumin binding residue or pegylated ina position selected from a position equivalent to position 18, 20, 23,30, 31, 34, 36, 37, 38, or 39 of GLP-1(7-37) (SEQ ID No: 1).

-   18. The GLP-1 derivative according to embodiment 17,    wherein    (Xaa₇-Xaa₈) is (L-histidine-Aib), or (desamino-histidine-alanine);    Xaa₉ is Glu;    Xaa₁₆ is Val;    Xaa₁₈ is Ser;    Xaa₂₀ is Leu, or Lys;    Xaa₂₃ is Gln;    Xaa₂₄ is Ala;    Xaa₂₅ is Ala, or Val;    Xaa₂₇ is Glu;    Xaa₂₈ is Phe, or a Phe derivative such as m-CF₃-Phe;    Xaa₃₀ is Ala, Glu, or Lys;    Xaa₃₁ is Trp, or Lys;    Xaa₃₃ is Val;    Xaa₃₄ is Lys, Dap, or Arg;    Xaa₃₅ is Gly, Arg, Lys, Aib, or absent;    Xaa₃₆ is Arg, Lys, or absent;    Xaa₃₇ is Gly, Lys, epsilon-amino-Lys, Pro, Arg, or absent;    Xaa₃₈ is Lys, Glu, Arg, or absent;    Xaa₃₉ is Lys, Arg, or absent;    Xaa₄₀ is Arg, or absent;    Xaa₄₁ is Arg, or absent; and    R is amide, or absent;    provided that if Xaa₃₇, Xaa₃₈, Xaa₃₉, or Xaa₄₀ is absent, then each    amino acid residue downstream is also absent;    and which is derivatised with an albumin binding residue or    pegylated in a position selected from a position equivalent to    position 18, 20, 23, 30, 31, 34, 36, 37, 38, or 39 of GLP-1(7-37)    (SEQ ID No: 1).-   19. A GLP-1 derivative, preferably according to any one of    embodiments 1-18, having the sequence of formula (II)

Formula (II) (SEQ ID No: 3)Xaa₇-Xaa₈-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Xaa₁₈-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Arg-Glu-Phe-Ile-Xaa₃₀-Xaa₃₁-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇- Xaa₃₈-Xaa₃₉-Xaa₄₀-Xaa₄₁-RwhereinXaa₇ is L-histidine, D-histidine, desamino-histidine, 2-amino-histidine,β-hydroxy-histidine, homohistidine, N^(α)-acetyl-histidine,β-fluoromethyl-histidine, α-methyl-histidine, 3-pyridylalanine,2-pyridylalanine, or 4-pyridylalanine;Xaa₈ is Ala, Gly, Val, Leu, Ile, Lys, Aib, (1-aminocyclopropyl)carboxylic acid, (1-aminocyclobutyl) carboxylic acid,(1-aminocyclopentyl) carboxylic acid, (1-aminocyclohexyl) carboxylicacid, (1-aminocycloheptyl) carboxylic acid, or (1-aminocyclooctyl)carboxylic acid;Xaa₁₈ is Ser, Lys, or Arg;Xaa₃₀ is Ala, Glu, Lys, or Arg;Xaa₃₁ is Lys, or Trp;Xaa₃₃ is Val or Lys;Xaa₃₄ is Lys, Glu, Dap, or Arg;Xaa₃₅ is Gly, Arg, Lys Aib, or absent;Xaa₃₆ is Arg, Lys, or absent;Xaa₃₇ is Gly, Aib, Lys, epsilon-amino-Lys, Pro, Arg, or absent;Xaa₃₈ is Lys, Glu, Arg, or absent;Xaa₃₉ is Lys, Arg, or absent;Xaa₄₀ is Arg, or absent;Xaa₄₁ is Arg, or absent; andR is amide, or is absent;provided that if Xaa₃₇, Xaa₃₈, Xaa₃₉, or Xaa₄₀ is absent, then eachamino acid residue downstream is also absent;and which is derivatised with an albumin binding residue or pegylated ina position selected from a position equivalent to position 18, 20, 23,30, 31, 34, 36, 37, 38, or 39 of GLP-1(7-37) (SEQ ID No: 1).

-   20. The GLP-1 derivative according to embodiment 19,    wherein    Xaa₇ is L-histidine, or desamino-histidine;    Xaa₈ is Ala, or Aib;    Xaa₁₈ is Ser;    Xaa₃₀ is Ala, Glu, or Lys;    Xaa₃₁ is Lys, or Trp;    Xaa₃₃ is Val;    Xaa₃₄ is Lys, Dap, or Arg;    Xaa₃₅ is Gly, Arg, Lys Aib, or absent;    Xaa₃₆ is Arg, Lys, or absent;    Xaa₃₇ is Gly, Lys, epsilon-amino-Lys, Pro, Arg, or absent;    Xaa₃₈ is Lys, Glu, Arg, or absent;    Xaa₃₉ is Lys, Arg, or absent;    Xaa₄₀ is Arg, or absent;    Xaa₄₁ is Arg, or absent; and    R is amide, or is absent;    provided that if Xaa₃₇, Xaa₃₈, Xaa₃₉, or Xaa₄₀ is absent, then each    amino acid residue downstream is also absent;    and which is derivatised with an albumin binding residue or    pegylated in a position selected from a position equivalent to    position 18, 20, 23, 30, 31, 34, 36, 37, 38, or 39 of GLP-1(7-37)    (SEQ ID No: 1).-   21. The GLP-1 derivative according to any one of embodiments 1-20    which is derivatised in one position.-   22. The GLP-1 derivative according to any one of embodiments 1-20    which is derivatised in one or more positions, preferably in two    position, in three positions, or in four positions.-   23. The GLP-1 derivative according to any one of embodiments 1-22    which is derivatised in i) position 18, ii) position 20, iii)    position 23, iv) position 30, v) position 31, vi) position 34, vii)    position 36, viii) position 37, ix) position 38, and/or x) position    39.-   24. The GLP-1 derivative according to any one of embodiments 1-23    which is derivatised in i) position 20, ii) position 30, iii)    position 31, iv) position 36, v) position 37, vi) position 38,    or vii) position 39.-   25. The GLP-1 derivative according to any one of embodiments 1-24    which is PEGylated.-   26. The GLP-1 derivative according to any one of embodiments 1-24    wherein the albumin binding residue is selected from i) straight    chain alkyl, preferably C15 alkyl; ii) acyl of the formula    CH₃(CH₂)_(r)CO—, wherein preferably r is 14 or 16.-   27. The GLP-1 derivative according to any one of embodiments 1-24    and 26 which includes a linker between the albumin binding residue    and GLP-1 moiety, wherein the linker comprises, preferably is,    i) one or more alkylene glycol units such as 1-10, preferably 5-8,    more preferably 6;    ii) a compound C of formula X wherein c is 0 and b is 2;    iii) a compound of formula XVI wherein q is 1 or 2; or    iv) any combination of i)-iii), such as a combination of ii) and    iii).-   28. A GLP-1 derivative, preferably according to any one of the    embodiments 1-27, wherein at least one amino acid residue is    derivatised with A-B-C-D-    wherein A- is selected from the group consisting of

wherein the tetrazole ring is optionally N-substituted, andwherein n is selected from the group consisting of 14, 15, 16 17, 18 and19, p is selected from the group consisting of 10, 11, 12, 13 and 14,and d is selected from the group consisting of 0, 1, 2, 3, 4 and 5,—B— is selected from the group consisting of

wherein x is selected from the group consisting of 0, 1, 2, 3 and 4, andy is selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11 and 12,—C— is selected from the group consisting of

wherein b and e are each independently selected from the groupconsisting of 0, 1 and 2, and c and f are each independently selectedfrom the group consisting of 0, 1 and 2 with the proviso that b is 1 or2 when c is 0, or b is 0 when c is 1 or 2, and e is 1 or 2 when f is 0,or e is 0 when f is 1 or 2, and-D- is attached to said amino acid residue and is a linker.

-   29. The GLP-1 derivative according to any one of embodiments 1-28,    wherein the derivatised amino acid residue is lysine, which is    preferably derivatised via the epsilon amino group.-   30. A GLP-derivative, preferably according to any one of embodiments    1-29, which is selected from the following:-   N-epsilon37{2-[2-(2-{2-[2-((R)-3-carboxy-3-{[1-(19-carboxynonadecanoyl)piperidine-4-carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl    [desaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1(7-37)amide;-   N-epsilon37{2-[2-(2-{2-[2-((S)-3-carboxy-3-{[1-(19-carboxynonadecanoyl)piperidine-4-carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl    [Aib8,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide;-   N-epsilon37-[2-(2-[2-(2-[2-(2-((R)-3-[1-(17-Carboxyheptadecanoyl)piperidin-4-ylcarbonylamino]3-carboxypropionylamino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][DesaminoHis    7,Glu22,Arg26,Arg34,Phe(m-CF3)28]GLP-1-(7-37)amide;-   N-epsilon30{2-[2-(2-{2-[2-((S)-3-carboxy-3-{[1-(19-carboxynonadecanoyl)piperidine-4-carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl    [Aib8,Glu22,Arg26,Lys30]GLP-1-(7-37);-   N-epsilon31{2-[2-(2-{2-[2-((S)-3-carboxy-3-{[1-(19-carboxynonadecanoyl)piperidine-4-carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl    [Aib8,Glu22,Arg26,Lys31]GLP-1-(7-37);-   N-epsilon31-(2-{2-[2-(2-{2-[2-((S)-3-Carboxy-3-{[1-(19-carboxy-nonadecanoyl)piperidine-4-carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl)[Aib    8,Glu22,Arg26,Lys31,Arg34]GLP-1-(7-37);-   N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxy-nonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide;-   N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxy-nonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide;-   N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxy-nonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37);-   N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxy-nonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg26,Glu30,Arg34,Lys37]GLP-1-(7-37);-   N-epsilon20-[2-(2-{2-[(S)-4-Carboxy-4-((S)-4-carboxy-4-{12-[4-(16-(1H-tetrazol-5-yl)hexadecanoylsulfamoyl)butyrylamino]dodecanoylamino}butyrylamino)butyrylamino]ethoxy}ethoxy)acetyl][Aib8,Lys20,Glu22,Arg26,Glu30,Pro37]GLP-1-(7-37)amide;-   N-epsilon37-[2-(2-{2-[(S)-4-Carboxy-4-((S)-4-carboxy-4-{12-[4-(16-(1H-tetrazol-5-yl)hexadecanoylsulfamoyl)butyrylamino]dodecanoylamino}butyrylamino)butyrylamino]ethoxy}ethoxy)acetyl][Aib8,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide;-   N-epsilon37-[2-(2-{2-[(S)-4-Carboxy-4-((S)-4-carboxy-4-{12-[4-(16-(1H-tetrazol-5-yl)hexadecanoylsulfamoyl)butyrylamino]dodecanoylamino}butyrylamino)butyrylamino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide;-   [Aib8,Glu22,Arg26,Glu30,Pro37]GLP-1-(7-37)Lys    [2-(2-{2-[4-Carboxy-4-(4-carboxy-4-{4-[4-(16-1H-tetrazol-5-yl-hexadecanoylsulfamoyl)butyrylamino]butyrylamino}butyrylamino)butyrylamino]ethoxy}ethoxy)acetyl];-   N-epsilon37    (Polyethyleneglycol2000)[DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1    (7-37) amide;-   N-epsilon37    (3-((2-(2-(2-(2-(2-Hexadecyloxyethoxy)ethoxy)ethoxy)ethoxy)ethoxy))propionyl)[DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1(7-37)-amide;-   N-epsilon37-{2-(2-(2-(2-[2-(2-(4-(hexadecanoylamino)-4-carboxybutyrylamino)ethoxy)ethoxy]acetyl)ethoxy)ethoxy)acetyl)}-[desaminoHis7,Glu22,Arg26,    Glu30,Arg34,Lys37] (GLP-1-(7-37)amide-   N-epsilon37-{2-(2-(2-(2-[2-(2-(4-(hexadecanoylamino)-4-carboxybutyrylamino)ethoxy)ethoxy]acetyl)ethoxy)ethoxy)acetyl)}-[desaminoHis7,Glu22,    Arg26,Arg34,Lys 37] (GLP-1-(7-37)amide;-   N-epsilon37-(2-(2-(2-(2-(2-(2-(2-(2-(2-(Octadecanoylamino)ethoxy)ethoxy)acetylamino)ethoxy)ethoxy)acetylamino)ethoxy)ethoxy)acetyl)[desaminoHis7,Glu22,Arg26,Arg34,Lys37]    GLP-1 (7-37)amide-   N-epsilon36-(2-(2-(2-((2-[2-(2-(17-carboxyheptadecanoylamino)ethoxy)ethoxy]acetylamino)ethoxy)ethoxy)acetyl)    [Aib8,Glu22,Arg26,Glu30,Lys36] GLP-1-(7-37)Glu-amide;-   N-epsilon37-[4-(16-(1H-Tetrazol-5-yl)hexadecanoylsulfamoyl)butyryl][DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide;-   N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(19-carboxynonadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37);-   N-epsilon31-[2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Glu22,Arg26,Lys31]GLP-1-(7-37);-   N-epsilon20-(2-{2-[2-(2-{2-[2-((S)-4-Carboxy-4-hexadecanoylamino-butyrylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}-acetyl)[Aib8,Lys20,Glu22,Arg26,Glu30,Pro37]GLP-1-(7-37)amide;-   N-epsilon37-(2-{2-[2-((S)-4-Carboxy-4-{(S)-4-carboxy-4-[(S)-4-carboxy-4-(19-carboxy-nonadecanoylamino)butyrylamino]butyrylamino}butyrylamino)ethoxy]ethoxy}acetyl)[DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37);-   N-epsilon37-{2-[2-(2-{(S)-4-[(S)-4-(12-{4-[16-(2-tert-Butyl-2H-tetrazol-5-yl)-hexadecanoylsulfamoyl]butyrylamino}dodecanoylamino)-4-carboxybutyrylamino]-4-carboxybutyrylamino}ethoxy)ethoxy]acetyl}[DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1    (7-37);-   N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxy-heptadecanoylamino)-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl][Aib8,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37).-   N-alpha37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxy-heptadecanoylamino)-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl][Aib8,Glu22,Arg26,Arg34,epsilon-Lys37]GLP-1-(7-37)peptide;-   N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxy-heptadecanoylamino)-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl][desaminoHis7,Glu22,Arg26,Arg34,Lys37]    GLP-1-(7-37);-   N-epsilon36-[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(15-carboxy-pentadecanoylamino)-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl][desaminoHis7,    Glu22,Arg26,Glu30,Arg34,Lys36]GLP-1-(7-37)-Glu-Lys peptide;-   [desaminoHis7,Glu22,Arg26,Glu30,Arg34]GLP-1-(7-37)-Glu-Lys(2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxy-heptadecanoylamino)-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl)peptide;-   N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37);-   N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxy-heptadecanoylamino)-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl]-[Aib8,Glu22,    Arg26,Arg34,Aib35,Lys37]GLP-1-(7-37);-   N-epsilon31-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Glu22,Val25,Arg26,Lys31,Arg34,Arg35,Arg37]GLP-1-(7-37);-   N-epsilon31{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-[Aib8,Glu22,Val25,Arg26,Lys31,Arg34,Arg35,Arg37)]GLP-1(7-37)yl    [Arg39,Arg40,Arg41];-   N-epsilon31-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Glu22,Val25,Arg26,Lys31,Lys35,Lys36]GLP-1-(7-36)    amide;-   N-epsilon31-{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-N-beta34-(2-(bis-carboxymethylamino)acetyl)[Aib8,Glu22,Val25,Arg26,Lys31,Dap34]GLP-1(7-34)    amide; and-   N-epsilon-37-[(S)-4-carboxy-4-(2-{2-[2-(2-{2-[2-(17-carboxyheptadecanoylamino)    ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetylamino)butyryl][Aib8,Glu22,Arg26,34,Lys37]    GLP-1 (7-37).-   31. A pharmaceutical composition comprising a derivative according    to any one of embodiments 1-30 or a pharmaceutically acceptable    salt, amide, alkyl, or ester or the like thereof, and a    pharmaceutically acceptable excipient.-   32. A derivative according to any one of embodiments 1-30, or a    pharmaceutical composition according to embodiment 31, for use as a    medicament.-   33. A derivative according to any one of embodiments 1-30, or a    pharmaceutical composition according to embodiment 31, for use in    the treatment or prevention of hyperglycemia, type 2 diabetes,    impaired glucose tolerance, type 1 diabetes, obesity, hypertension,    syndrome X, dyslipidemia, cognitive disorders, atherosclerosis,    myocardial infarction, coronary heart disease and other    cardiovascular disorders, stroke, inflammatory bowel syndrome,    dyspepsia and gastric ulcers.-   34. The derivative or pharmaceutical composition according to    embodiment 33, for use in the treatment or prevention of type 2    diabetes.-   35. Use of a derivative according to any one of embodiments 1-30, or    a pharmaceutical composition according to embodiment 31, in the    manufacture of a medicament for use in the treatment or prevention    of hyperglycemia, type 2 diabetes, impaired glucose tolerance, type    1 diabetes, obesity, hypertension, syndrome X, dyslipidemia,    cognitive disorders, atherosclerosis, myocardial infarction,    coronary heart disease and other cardiovascular disorders, stroke,    inflammatory bowel syndrome, dyspepsia and gastric ulcers.-   36. The use according to embodiment 35 for the treatment or    prevention of type 2 diabetes.-   37. A method of treating or preventing hyperglycemia, type 2    diabetes, impaired glucose tolerance, type 1 diabetes, obesity,    hypertension, syndrome X, dyslipidemia, cognitive disorders,    atherosclerosis, myocardial infarction, coronary heart disease and    other cardiovascular disorders, stroke, inflammatory bowel syndrome,    dyspepsia and gastric ulcers by administering a pharmaceutically    active amount of a derivative according to any one of embodiments    1-30, or a pharmaceutical composition according to embodiment 31.-   38. The method according to embodiment 37 for treating or preventing    type 2 diabetes.

All references, including publications, patent applications and patents,cited herein are hereby incorporated by reference to the same extent asif each reference was individually and specifically indicated to beincorporated by reference and was set forth in its entirety herein.

All headings and sub-headings are used herein for convenience only andshould not be construed as limiting the invention in any way,

Any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

The terms “a” and “an” and “the” and similar referents as used in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context.

Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. Unless otherwise stated, all exact valuesprovided herein are representative of corresponding approximate values(e.g., all exact exemplary values provided with respect to a particularfactor or measurement can be considered to also pro-vide a correspondingapproximate measurement, modified by “about,” where appropriate).

All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext.

The use of any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise indicated. No language in the specification should beconstrued as indicating any element is essential to the practice of theinvention unless as much is explicitly stated.

The citation and incorporation of patent documents herein is done forconvenience only and does not reflect any view of the validity,patentability and/or enforceability of such patent documents,

The description herein of any aspect or embodiment of the inventionusing terms such as “comprising”, “having”, “including” or “containing”with reference to an element or elements is intended to provide supportfor a similar aspect or embodiment of the invention that “consists of”,“consists essentially of”, or “substantially comprises” that particularelement or elements, unless otherwise stated or clearly contradicted bycontext (e.g., a formulation described herein as comprising a particularelement should be understood as also describing a formulation consistingof that element, unless otherwise stated or clearly contradicted bycontext).

This invention includes all modifications and equivalents of the subjectmatter recited in the embodiments, aspects or claims presented herein tothe maximum extent permitted by applicable law.

The present invention is further illustrated in the followingrepresentative methods and examples which are, however, not intended tolimit the scope of the invention in any way.

The features disclosed in the foregoing description and in the followingexamples may, both separately and in any combination thereof, bematerial for realising the invention in diverse forms thereof.

EXAMPLES Abbreviations Used

r.t: Room temperature

DIPEA: diisopropylethylamine

H₂O: water

CH₃CN: acetonitrile

DMF: NN dimethylformamide

HBTU: 2-(1H-Benzotriazol-1-yl-)-1,1,3,3 tetramethyluroniumhexafluorophosphate

Fmoc: 9 H-fluoren-9-ylmethoxycarbonyl

Boc: tert butyloxycarbonyl

OtBu: tert butyl ester

tBu: tert butyl

Trt: triphenylmethyl

Pmc: 2,2,5,7,8-Pentamethyl-chroman-6-sulfonyl

Dde: 1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)ethyl

ivDde: 1-(4,4-Dimethyl-2,6-dioxocyclohexylidene)-3-methylbutyl

Mtt: 4-methyltrityl

Mmt: 4-methoxytrityl

DCM: dichloromethane

TIS: triisopropylsilane)

TFA: trifluoroacetic acid

Et₂O: diethylether

NMP: 1-Methyl-pyrrolidin-2-one

DIPEA: Diisopropylethylamine

HOAt: 1-Hydroxy-7-azabenzotriazole

HOBt: 1-Hydroxybenzotriazole

DIC: Diisopropylcarbodiimide

MW: Molecular weight

A: Synthesis of Resin Bound Peptide

SPPS Method A.

The protected peptidyl resin was synthesized according to the Fmocstrategy on an Applied Biosystems 433 peptide synthesizer in 0.25 mmolor 1.0 mmol scale using the manufacturer supplied FastMoc UV protocolswhich employ HBTU (2-(1H-Benzotriazol-1-yl-)-1,1,3,3 tetramethyluroniumhexafluorophosphate) or HATU(O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate) mediated couplings in NMP(N-methylpyrrolidone), andUV monitoring of the deprotection of the Fmoc protection group. Thestarting resin used for the synthesis of the peptide amides wasRink-Amide resin and either Wang or chlorotrityl resin was used forpeptides with a carboxy C-terminal. The protected amino acid derivativesused were standard Fmoc-amino acids (supplied from e.g. Anaspec, orNovabiochem) supplied in preweighed cartridges suitable for the ABI433Asynthesizer with the exception of unnatural aminoacids such asFmoc-Aib-OH (Fmoc-aminoisobutyric acid). The N terminal amino acid wasBoc protected at the alpha amino group (e.g. Boc-His(Boc)OH was used forpeptides with His at the N-terminal). The epsilon amino group of lysinein position 26 was either protected with Mtt, Mmt, Dde, ivDde, or Boc,depending on the route for attachment of the albumin binding moiety andspacer. The synthesis of the peptides may in some cases be improved bythe use of dipeptides protected on the dipeptide amide bond with a groupthat can be cleaved under acidic conditions such but not limited to2-Fmoc-oxy-4-methoxybenzyl or 2,4,6-trimethoxybenzyl. In cases where aserine or a threonine is present in the peptide, the use ofpseudoproline dipeptides may be used (see e.g. catalogue fromNovobiochem 2002/2003 or newer version, or W. R. Sampson (1999), J. Pep.Sci. 5, 403.

SPPS Method B:

One alternative method (method B) of peptide synthesis was by Fmocchemistry on a microwave-based Liberty peptide synthesizer (CEM Corp.,North Carolina). The resin was Tentagel S RAM with a loading of 0.24mmol/g. The coupling chemistry was DIC/HOAt in NMP using amino acidsolutions of 0.3 M in NMP and a molar excess of 8-10 fold. Couplingconditions was 5 minutes at up to 70° C. Deprotection was with 5%piperidine in NMP at up to 70° C. When a chemical modification of alysine side chain was desired, the lysine was incorporated as Lys(Mtt).The Mtt group was removed by suspending the resin in neathexafluoroisopropanol for 20 minutes followed by washing with DCM andNMP. The chemical modification of the lysine was performed either bymanual synthesis or by one or more automated steps on the Libertyfollowed by a manual coupling. Another method of peptide synthesis wasby Fmoc chemistry on an ABI 433 with HBTU coupling. After synthesis theresin was washed with DCM and dried, and the peptide was cleaved fromthe resin by a 2 hour treatment with TFA/TIS/water (92.5/5/2.5) followedby precipitation with diethylether. the peptide was redissolved in 30%acetic acid or similar solvent and purified by standard RP-HPLC on a C18column using acetonitrile/TFA. The identity of the peptide was confirmedby MALDI-MS.

SPPS Method C

The protected peptidyl resin was synthesized according to the Fmocstrategy on an Advanced ChemTech Synthesiser (APEX 348) 0.25 mmol scaleusing the manufacturer supplied protocols which employ DIC(dicyclohexylcarbodiimide) and HOBt (1-Hydroxybenzotriazole) mediatedcouplings in NMP(N-methylpyrrolidone. The starting resin used for thesynthesis of the peptide amides was Rink-Amide resin and either Wang orchlorotrityl resin was used for peptides with a carboxy C-terminal. Theprotected amino acid derivatives used were standard Fmoc-amino acids(supplied from e.g. Anaspec, or Novabiochem. The N terminal amino acidwas Boc protected at the alpha amino group (e.g. Boc-His(Boc)OH was usedfor peptides with His at the N-terminal). The epsilon amino group oflysine in position 26 was either protected with Mtt, Mmt, Dde, ivDde, orBoc, depending on the route for attachment of the albumin binding moietyand spacer. The synthesis of the peptides may in some cases be improvedby the use of dipeptides, e.g., pseudoprolines from Novabiochem,Fmoc-Ser(tbu)-ΨSer(Me,Me)-OH, see e.g. catalogue from Novobiochem2002/2003 or newer version, or W. R. Sampson (1999), J. Pep. Sci. 5, 403

Procedure for Removal of ivDde or Dde-Protection.

The resin (0.25 mmol) was placed in a manual shaker/filtration apparatusand treated with 2% hydrazine in N-methylpyrrolidone (20 ml, 2×12 min)to remove the Dde or ivDde group and wash with N-methylpyrrolidone (4×20ml).

Procedure for Removal of Mtt or Mmt-Protection.

The resin (0.25 mmol) was placed in a manual shaker/filtration apparatusand treated with 2% TFA and 2-3% TIS in DCM (20 ml, 5-10 min repeated6-12 times) to remove the Mtt or Mmt group and wash with DCM (2×20 ml),10% MeOH and 5% DIPEA in DCM (2×20 ml) and N-methylpyrrolidone (4×20ml).

Alternative Procedure for Removal of Mtt-Protection:

The resin was placed in a syringe and treated with hexafluoroisopropanolfor 2×10 min to remove the Mtt group. The resin was then washed with DCMand NMP as described above.

Procedure for Attachment of Sidechains to Lysine Residue.

The albumin binding residue (B—U— sidechain of formula I) can beattached to the peptide either by acylation to resin bound peptide oracylation in solution to the unprotected peptide using standardacylating reagent such as but not limited to DIC, HOBt/DIC, HOAt/DIC, orHBTU.

Attachment to Resin Bound Peptide:

Route I

Activated (active ester or symmetric anhydride) albumin binding residue(A-B)-sidechain of formula I) such as octadecanedioic acidmono-(2,5-dioxo-pyrrolidin-1-yl) ester (Ebashi et al. EP511600, 4 molarequivalents relative to resin bound peptide) was dissolved in NMP (25mL), added to the resin and shaken overnight at room temperature. Thereaction mixture was filtered and the resin was washed extensively withNMP, dichloromethane, 2-propanol, methanol and diethyl ether.

Route II

The albumin binding residue (A-(B)- sidechain of formula I) wasdissolved in N-methyl pyrrolidone/methylene chloride (1:1, 10 ml). Theactivating reagent such as hydroxybenzotriazole (HOBt) (4 molarequivalents relative to resin) and diisopropylcarbodiimide (4 molarequivalents relative to resin) was added and the solution was stirredfor 15 min. The solution was added to the resin anddiisopropylethylamine (4 molar equivalents relative to resin) was added.The resin was shaken 2 to 24 hours at room temperature. The resin waswashed with N-methyl pyrrolidone (2×20 ml),N-methylpyrrolidone/Methylene chloride (1:1) (2×20 ml) and methylenechloride (2×20 ml).

Route III

Activated (active ester or symmetric anhydride) albumin binding residue(A-B- sidechain of formula I) such as octadecanedioic acidmono-(2,5-dioxo-pyrrolidin-1-yl) ester (Ebashi et al. EP511600, 1-1.5molar equivalents relative to the peptide was dissolved in an organicsolvent such as acetonitrile, THF, DMF, DMSO or in a mixture ofwater/organic solvent (1-2 ml) and added to a solution of the peptide inwater (10-20 ml) together with 10 molar equivalents of DIPEA. In case ofprotecting groups on the albumin binding residue such as tert.-butyl,the reaction mixture was lyophilized 0/N and the isolated crude peptidedeprotected afterwards—in case of a tert-butyl group the peptide wasdissolved in a mixture of trifluoroacetic acid, water andtriisopropylsilane (90:5:5). After for 30 min the mixture was,evaporated in vacuo and the finale peptide purified by preparative HPLC.

Procedure for Removal of Fmoc-Protection:

The resin (0.25 mmol) was placed in a filter flask in a manual shakingapparatus and treated with N-methylpyrrolidone/methylene chloride (1:1)(2×20 ml) and with N-methylpyrrolidone (1×20 ml), a solution of 20%piperidine in N-methylpyrrolidone (3×20 ml, 10 min each). The resin waswashed with N-methylpyrrolidone (2×20 ml), N-methylpyrrolidone/Methylenechloride (1:1) (2×20 ml) and methylene chloride (2×20 ml).

Procedure for Cleaving the Peptide Off the Resin:

The peptide was cleaved from the resin by stirring for 180 min at roomtemperature with a mixture of trifluoroacetic acid, water andtriisopropylsilane (95:2.5:2.5 to 92:4:4). The cleavage mixture wasfiltered and the filtrate was concentrated to an oil by a stream ofnitrogen. The crude peptide was precipitated from this oil with 45 mldiethyl ether and washed 1 to 3 times with 45 ml diethyl ether.

Purification:

The crude peptide was purified by semipreparative HPLC on a 20 mm×250 mmcolumn packed with either 5μ or 7μ C-18 silica. Depending on the peptideone or two purification systems were used.

TFA: After drying the crude peptide was dissolved in 5 ml 50% aceticacid H₂O and diluted to 20 ml with H₂O and injected on the column whichthen was eluted with a gradient of 40-60% CH₃CN in 0.1% TFA 10 ml/minduring 50 min at 40° C. The peptide containing fractions were collected.The purified peptide was lyophilized after dilution of the eluate withwater.

Ammonium sulphate: The column was equilibrated with 40% CH₃CN in 0.05M(NH₄)₂SO₄, which was adjusted to pH 2.5 with concentrated H₂SO₄. Afterdrying the crude peptide was dissolved in 5 ml 50% acetic acid H₂O anddiluted to 20 ml with H₂O and injected on the column which then waseluted with a gradient of 40%-60% CH₃CN in 0.05M (NH₄)₂SO₄, pH 2.5 at 10ml/min during 50 min at 40° C. The peptide containing fractions werecollected and diluted with 3 volumes of H₂O and passed through aSep-Pak® C18 cartridge (Waters part. #:51910) which has beenequilibrated with 0.1% TFA. It was then eluted with 70% CH₃CN containing0.1% TFA and the purified peptide was isolated by lyophilisation afterdilution of the eluate with water.

The final product obtained was characterised by analytical RP-HPLC(retention time) and by LCMS

The RP-HPLC analysis may be performed using UV detection at 214 nm ande.g. a Vydac 218TP54 4.6 mm×250 mm 5μ C-18 silica column (TheSeparations Group, Hesperia, USA) and eluted at e.g. 1 ml/min at 42° C.Most often one of following specific conditions were used:

Method 03_A1_(—)1

HPLC (Method 03_A1_(—)1): The RP-analysis was performed using a Waters2690 systems fitted with a Waters 996 diode array detector. UVdetections were collected at 214, 254, 276, and 301 nm on a 218TP54 4.6mm×250 mm 5μ C-18 silica column (The Seperations Group, Hesperia), whichwas eluted at 1 ml/min at 42° C. The column was equilibrated with 10% ofa 0.5 M ammonium sulfate, which was adjusted to pH 2.5 with 4M sulfuricacid. After injection, the sample was eluted by a gradient of 0% to 60%acetonitrile in the same aqueous buffer during 50 min.

Method 03_B1_(—)2

HPLC (Method 03_B1_(—)2): The RP-analysis was performed using a Waters2690 systems fitted with a Waters 996 diode array detector. UVdetections were collected at 214, 254, 276, and 301 nm on a Zorbax 300SBC-18 (4,5×150 mm, 50, which was eluted at 0.5 ml/min at 42° C. Thecolumn was equilibrated with an aqueous solution of TFA in water (0.1%).After injection, the sample was eluted by a gradient of 0% to 60%acetonitrile (+0.1% TFA) in an aqueous solution of TFA in water (0.1%)during 50 min.

Method 02_B1_(—)1

HPLC (Method 02_B1_(—)1): The RP-analyses was performed using a AllianceWaters 2695 system fitted with a Waters 2487 dualband detector. UVdetections at 214 nm and 254 nm were collected using a Vydac 218TP53,C18, 300 Å, 5 um, 3.2 mm×250 mm column, 42° C. Eluted with a lineargradient of 0-60% acetonitrile, 95-35% water and 5% trifluoroacetic acid(1.0%) in water over 50 minutes at a flow-rate of 0.50 ml/min.

Method 01_B4_(—)2

HPLC (Method 01_B4_(—)2): RP-analyses was performed using a Waters 600Ssystem fitted with a Waters 996 diode array detector. UV detections at214 nm and 254 nm were collected using a Symmetry300 C18, 5 um, 3.9mm×150 mm column, 42° C. Eluted with a linear gradient of 5-95%acetonitrile, 90-0% water, and 5% trifluoroacetic acid (1.0%) in waterover 15 minutes at a flow-rate of 1.0 min/min.

Method 02_B4_(—)4

HPLC (Method 02_B4_(—)4): The RP-analyses was performed using a AllianceWaters 2695 system fitted with a Waters 2487 dualband detector. UVdetections at 214 nm and 254 nm were collected using a Symmetry300 C18,5 um, 3.9 mm×150 mm column, 42° C. Eluted with a linear gradient of5-95% acetonitrile, 90-0% water, and 5% trifluoroacetic acid (1.0%) inwater over 15 minutes at a flow-rate of 1.0 min/min.

Method 02_B6_(—)1

HPLC (Method 02_B6_(—)1): The RP-analyses was performed using a AllianceWaters 2695 system fitted with a Waters 2487 dualband detector. UVdetections at 214 nm and 254 nm were collected using a Vydac 218TP53,C18, 300 Å, 5 um, 3.2 mm×250 mm column, 42° C. Eluted with a lineargradient of 0-90% acetonitrile, 95-5% water, and 5% trifluoroacetic acid(1.0%) in water over 50 minutes at a flow-rate of 0.50 ml/min.

Method 03_B6_(—)1

HPLC (Method 03_B6_(—)1): The RP-analysis was performed using a Waters2690 systems fitted with a Waters 996 diode array detector. UVdetections were collected at 214, 254, 276, and 301 nm on a 218TP54 4.6mm×250 mm 5μ C-18 silica column (The Seperations Group, Hesperia), whichwas eluted at 1 ml/min at 42° C. The column was equilibrated with 5%acetonitrile (+0.1% TFA) in an aqueous solution of TFA in water (0.1%).After injection, the sample was eluted by a gradient of 0% to 90%acetonitrile (+0.1% TFA) in an aqueous solution of TFA in water (0.1%)during 50 min.

Alternatively a preparative gradient elution can be performed asindicated above and the percentage of acetonitrile where the compoundelutes is noted. Identity is confirmed by MALDI.

The following instrumentation was used:

LCMS was performed on a setup consisting of Sciex API 100 Singlequadrupole mass spectrometer, Perkin Elmer Series 200 Quard pump, PerkinElmer Series 200 autosampler, Applied Biosystems 785A UV detector, Sedex75 evaporative light scattering detector

The instrument control and data acquisition were done by the SciexSample control software running on a Windows 2000 computer.

The HPLC pump is connected to two eluent reservoirs containing:

A: 0.05% Trifluoro acetic acid in water

B: 0.05% Trifluoro acetic acid in acetonitrile

The analysis is performed at room temperature by injecting anappropriate volume of the sample (preferably 20 μl) onto the columnwhich is eluted with a gradient of acetonitrile.

The HPLC conditions, detector settings and mass spectrometer settingsused are giving in the following table.

Column: Waters Xterra MS C-18 × 3 mm id 5 μm Gradient: 5%-90%acetonitrile linear during 7.5 min at 1.5 ml/min Detection: 210 nm(analogue output from DAD) ELS (analogue output from ELS), 40° C. MSionisation mode API-ES

Alternatively LCMS was performed on a setup consisting of HewlettPackard series 1100 G1312A Bin Pump, Hewlett Packard series 1100 Columncompartment, Hewlett Packard series 1100 G1315A DAD diode arraydetector, Hewlett Packard series 1100 MSD and Sedere 75 EvaporativeLight Scattering detector controlled by HP Chemstation software. TheHPLC pump is connected to two eluent reservoirs containing:

A: 10 mM NH₄OH in water

B: 10 mM NH₄OH in 90% acetonitrile

The analysis was performed at 23° C. by injecting an appropriate volumeof the sample (preferably 20 μl) onto the column which is eluted with agradient of A and B.

The HPLC conditions, detector settings and mass spectrometer settingsused are giving in the following table.

Column Waters Xterra MS C-18 × 3 mm id 5 μm Gradient 5%-100%acetonitrile linear during 6.5 min at 1.5 ml/min Detection 210 nm(analogue output from DAD) ELS (analogue output from ELS) MS ionisationmode API-ES. Scan 100-1000 amu step 0.1 amuMALDI-MS:

Molecular weights of the peptides were determined using matrix-assistedlaser desorption time of flight mass spectroscopy (MALDI-MS), recordedon a Microflex (Bruker). A matrix of α-cyano-4-hydroxy cinnamic acid wasused.

Analytical HPLC Conditions (Method I):

Equilibration of the column (Xterra TM MS C18, 5 um, 4,6×150 mm Column,P7N 186 000490) with 0.1% TFA/H₂O and elution by a gradient of 0%CH₃CN/0.1% TFA/H₂O to 60% CH₃CN/0.1% TFA/H₂O during 25 min followed by agradient from 60% to 100% over 5 min.

In the examples of this invention the nomenclature and structurallygraphics is meant as:

One letter symbols for the natural amino acids are used, e.g. HisL-histidine, A is L-alanine ect. Three letter abbreviations for aminoacids may also be used, e.g. His is L-histidine, Ala is L-alanine etc.For non natural amino acids three letter abbreviations are used, such asAib for aminoisobutyric acid. The position of the amino acids may eitherbe indicated with a number in superscript after the amino acid symbolssuch as Lys³⁷, or as Lys37. The N-terminal amino group may be symbolisedeither as NH₂ or as H. The C-terminal carboxylic group may be symbolisedeither as —OH or as —COOH. The C-terminal amide group is symbolised as—NH₂

The sub-structures

both mean His-Aib-Glu-Gly-Thr-Phe.

The epsilon amino group of Lysine may be described either as the greeksymbol ε or spelled “epsilon”.

The structures in the examples below are in several cases a combinationof one letter symbols for the naturally amino acids combined with thethree letter abbreviation Aib for aminoisobutyric acid. In several casessome of the amino acids are shown in expanded full structure. Thuslysine that has been derivatised may be shown as the expanded fullstructures in example 1 where the lysine in position 37 is expanded. Thenitrogen (with indicated H) between arginine in position 36 and theexpanded lysine in position 37 is thus the nitrogen of the peptide bondconnecting the two amino acids in example 1.

According to the procedure above, the following derivatives wereprepared as non-limiting examples of the invention:

Example 1N-epsilon37{2-[2-(2-{2-[2-((R)-3-carboxy-3-{[1-(19-carboxynonadecanoyl)piperidine-4-carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl[desaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1(7-37)amide

Preparation method: AHPLC method B6:RT=35.49 minLCMS: m/z=1096.2 (M+3H)³⁺Calculated MW=4380.0

Example 2N-epsilon37{2-[2-(2-{2-[2-((S)-3-carboxy-3-{[1-(19-carboxynonadecanoyl)piperidine-4-carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl[Aib8,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide

Preparation method: BThe peptide was eluted at 66% acetonitrile.Structure confirmed by MALDI-MSCalculated MW=4409.1

Example 3N-epsilon37-[2-(2-[2-(2-[2-(2-((R)-3-[1-(17-Carboxyheptadecanoyl)piperidin-4-ylcarbonylamino]3-carboxypropionylamino)ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][DesaminoHis7,Glu22,Arg26,Arg34,Phe(m-CF3)28]GLP-1-(7-37)amide

Preparation method: AHPLC (method B):RT=11.92 minLCMS: m/z=1474.8 (M+3H)³⁺Calculated MW=4420.0

Example 4N-epsilon30{2-[2-(2-{2-[2-((S)-3-carboxy-3-{[1-(19-carboxynonadecanoyl)piperidine-4-carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl[Aib8,Glu22,Arg26,Lys30]GLP-1-(7-37)

Preparation method: Method A except that the peptide was prepared on anApex396 from Advanced Chemtech using a molar excess of 8-10 fold aminoacid, DIC and HOAt/HOBt (1:1) and the Mtt group was deprotected withhexafluoroisopropanol. The final product was characterized by analyticalHPLC and MALDI-MS.

HPLC method I:

RT=27.6 min

MALDI-MS: 4367.9

Example 5N-epsilon31{2-[2-(2-{2-[2-((S)-3-carboxy-3-{[1-(19-carboxynonadecanoyl)piperidine-4-carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl[Aib8,Glu22,Arg26,Lys31]GLP-1-(7-37)

Preparation method: Method A except that the peptide was prepared on anApex396 from Advanced Chemtech using a molar excess of 8-10 fold aminoacid, DIC and HOAt/HOBt (1:1) and the Mtt group was deprotected withhexafluoroisopropanol. The final product was characterized by analyticalHPLC and MALDI-MS.

HPLC method I:

RT=28.4 min

MALDI-MS: 4252.5

Example 6N-epsilon31-(2-{2-[2-(2-{2-[2-((S)-3-Carboxy-3-{[1-(19-carboxy-nonadecanoyl)piperidine-4-carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl)[Aib8,Glu22,Arg26,Lys31,Arg34]GLP-1-(7-37)

Preparation method: BThe peptide was eluted at 66% acetonitrile.Structure confirmed by MALDI-MSCalculated MW=4280.9

Example 7N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxy-nonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide

Preparation method: BThe peptide was eluted at 67% acetonitrile.Structure confirmed by MALDI-MSCalculated MW=4451.1

Example 8N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxy-nonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide

Preparation method: BThe peptide was eluted at 68% acetonitrile.Structure confirmed by MALDI-MSCalculated MW=4422.1

Example 9N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxy-nonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)

Preparation method: BThe peptide was eluted at 69% acetonitrile.Structure confirmed by MALDI-MSCalculated MW=4423.1

Example 10N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxy-nonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg26,Glu30,Arg34,Lys37]GLP-1-(7-37)

Preparation method: BThe peptide was eluted at 68% acetonitrile.Structure confirmed by MALDI-MSCalculated MW=4481.1

Example 11N-epsilon20-[2-(2-{2-[(S)-4-Carboxy-4-((S)-4-carboxy-4-{12-[4-(16-(1H-tetrazol-5-yl)hexadecanoylsulfamoyl)butyrylamino]dodecanoylamino}butyrylamino)butyrylamino]ethoxy}ethoxy)acetyl][Aib8,Lys20,Glu22,Arg26,Glu30,Pro37]GLP-1-(7-37)amide

Preparation method: BThe peptide was eluted at 62% acetonitrile.Structure confirmed by MALDI-MSCalculated MW=4638.3

Example 12N-epsilon37[2-(2-{2-[(S)-4-Carboxy-4-((S)-4-carboxy-4-{12-[4-(16-(1H-tetrazol-5-yl)hexadecanoylsulfamoyl)butyrylamino]dodecanoylamino}butyrylamino)butyrylamino]ethoxy}ethoxy)acetyl][Aib8,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide

Preparation method: BThe peptide was eluted at 69% acetonitrile.Structure confirmed by MALDI-MSCalculated MW=4624.3

Example 13N-epsilon37-[2-(2-{2-[(S)-4-Carboxy-4-((S)-4-carboxy-4-{12-[4-(16-(1H-tetrazol-5-yl)hexadecanoylsulfamoyl)butyrylamino]dodecanoylamino}butyrylamino)butyrylamino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide

Preparation method: BThe peptide was eluted at 65% acetonitrile.Structure confirmed by MALDI-MSCalculated MW=4595.3

Example 14 [Aib8,Glu22,Arg26,Glu30,Pro37]GLP-1-(7-37)Lys[2-(2-{2-[4-Carboxy-4-(4-carboxy-4-{4-[4-(16-1H-tetrazol-5-yl-hexadecanoylsulfamoyl)butyrylamino]butyrylamino}butyrylamino)butyrylamino]ethoxy}ethoxy)acetyl]

Preparation method: The peptide was prepared on an Apex396 from AdvancedChemtech and attachment of thioamide linker was achieved in two steps.4-sulfamoylbutyric acid was first coupled the resin using DIC andHOAt/HOBt (1:1). Then, 16-(1H-tetrazol-5-yl)hexadecanoic acid mixed withcarbonyldiimidazol in NMP was added to the resin followed by addition ofDBU. Otherwise is the same protocol described in the beginning of theexample section.

Calculated MW=4640.2

Example 15 N-epsilon37(Polyethyleneglycol2000)[DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1(7-37) amide

Preparation method: Route IIIHPLC (method A1)RT=40.88 minLCMS: m/z=Heterogenous but corresponding to stating material+an averageof 2000Calculated (M+H)⁺=5519

Example 16 N-epsilon37(3-((2-(2-(2-(2-(2-Hexadecyloxyethoxy)ethoxy)ethoxy)ethoxy)ethoxy))propionyl)[DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1(7-37)-amide

Preparation method: route IIIHPLC (method B6):RT=42.3 minLCMS: m/z=1353.3 (M+3H)³⁺Calculated (M+H)⁺=4055.7

Example 17N-epsilon37-{2-(2-(2-(2-[2-(2-(4-(hexadecanoylamino)-4-carboxybutyrylamino)ethoxy)ethoxy]acetyl)ethoxy)ethoxy)acetyl)}-[desaminoHis7,Glu22,Arg26,Glu30,Arg34,Lys37] (GLP-1-(7-37)amide

Preparation method: AHPLC (method B6):RT=36.1 minLCMS: m/z=1419.0 (M+3H)³⁺Calculated (M+H)⁺=4254.8

Example 18N-epsilon37-{2-(2-(2-(2-[2-(2-(4-(hexadecanoylamino)-4-carboxybutyrylamino)ethoxy)ethoxy]acetyl)ethoxy)ethoxy)acetyl)}-[desaminoHis7,Glu22,Arg26,Arg34,Lys 37] (GLP-1-(7-37)amide

Preparation method: AHPLC (method B6):RT=36.06 minLCMS: m/z=1399 (M+3H)³⁺Calculated (M+H)⁺=4196.8

Example 19 N-epsilon37-(2-(2-(2-(2-(2-(2-(2-(2-(2-(Octadecanoylamino)ethoxy)ethoxy)acetylamino)ethoxy)ethoxy)acetylamino)ethoxy)ethoxy)acetyl)[desaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1 (7-37)amide

Preparation method: AHPLC (method B6):RT=40.1 minLCMS: m/z=1414.6 (M+3H)³⁺Calculated (M+H)⁺=4240.9

Example 20 N-epsilon36-(2-(2-(2-((2-[2-(2-(17-carboxyheptadecanoylamino)ethoxy)ethoxy]acetylamino)ethoxy)ethoxy)acetyl)

[Aib8,Glu22,Arg26,Glu30,Lys36] GLP-1-(7-37)Glu-amide

Preparation method: BThe peptide was eluted at 68% acetonitrile.Structure confirmed by MALDI-MSCalculated MW=4069.6

Example 21N-epsilon37-[4-(16-(1H-Tetrazol-5-yl)hexadecanoylsulfamoyl)butyryl][DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide

Preparation method: BThe peptide was eluted at 64% acetonitrile.Structure confirmed by MALDI-MSCalculated MW=3994.6

Example 22N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(19-carboxynonadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)

Preparation method: BThe peptide was eluted at 67% acetonitrile.Structure confirmed by MALDI-MSCalculated MW=4283.9

Example 23N-epsilon31-[2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Glu22,Arg26,Lys31]GLP-1-(7-37)

Preparation method: Method A except that the peptide was prepared on anApex396 from Advanced Chemtech using a molar excess of 8-10 fold aminoacid, DIC and HOAt/HOBt (1:1) and the Mtt group was deprotected withhexafluoroisopropanol. The final product was characterized by analyticalHPLC and MALDI-MS.

HPLC method I:

RT=27.2 min

MALDI-MS: 4127.9

Example 24N-epsilon20-(2-{2-[2-(2-{2-[2-((S)-4-Carboxy-4-hexadecanoylamino-butyrylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}-acetyl)[Aib8,Lys20,Glu22,Arg26,Glu30,Pro37]GLP-1-(7-37)amide

Preparation method: BThe peptide was eluted at 66% acetonitrile.Structure confirmed by MALDI-MSCalculated MW=4239.8

Example 25N-epsilon37-(2-{2-[2-((S)-4-Carboxy-4-{(S)-4-carboxy-4-[(S)-4-carboxy-4-(19-carboxy-nonadecanoylamino)butyrylamino]butyrylamino}butyrylamino)ethoxy]ethoxy}acetyl)[DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)

Preparation method: BThe peptide was eluted at 65% acetonitrile.Structure confirmed by MALDI-MSCalculated MW=4397.0

Example 26N-epsilon37-{2-[2-(2-{(S)-4-[(S)-4-(12-{4-[16-(2-tert-Butyl-2H-tetrazol-5-yl)-hexadecanoylsulfamoyl]butyrylamino}dodecanoylamino)-4-carboxybutyrylamino]-4-carboxybutyrylamino}ethoxy)ethoxy]acetyl}[DesaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1 (7-37)

Preparation method BThe peptide was eluted at 74% acetonitrileStructure confirmed by MALDI-MSCalculated MW=4652.4

Example 27N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxy-heptadecanoylamino)-butyrylamino]-ethoxy}-ethoxy)-acetylamino]ethoxy}-ethoxy)-acetyl][Aib8,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)

Preparation analogous to SPPS Method B.HPLC method 02_B6_(—)1:RT=34.27 minLCMS: m/z=1072 (M+4H)⁴⁺Calculated (M)=4284.9

Example 28N-alpha37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxy-heptadecanoylamino)-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl][Aib8,Glu22,Arg26,Arg34,epsilon-Lys37]GLP-1-(7-37)peptide

Preparation analogous to SPPS Method B.HPLC method I_BDSB2:RT=10.19 minLCMS: m/z=1072 (M+4H)⁴⁺Calculated (M)=4284.9

Example 29N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxy-heptadecanoylamino)-butyrylamino]-ethoxy}-ethoxy)-acetylamino]ethoxy}-ethoxy)-acetyl][desaminoHis7,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)

Preparation analogous to SPPS Method B.HPLC method I_BDSB2:RT=9.96 minLCMS: m/z=1064 (M+4H)⁴⁺Calculated (M)=4255.8

Example 30N-epsilon36-[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(15-carboxy-pentadecanoylamino)-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl][desaminoHis7,Glu22, Arg26, Glu30, Arg34, Lys36]GLP-1-(7-37)-Glu-Lys peptide

Preparation analogous to SPPS Method B.HPLC method I_BDSB2:RT=6.40 minLCMS: m/z=1111 (M+4H)⁴⁺Calculated (M)=4444.0

Example 31 [desaminoHis7, Glu22, Arg26, Glu30,Arg34]GLP-1-(7-37)-Glu-Lys(2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxy-heptadecanoylamino)-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl)peptide

Preparation analogous to SPPS Method B.HPLC method I_BDSB2:RT=6.52 minLCMS: m/z=1119 (M+4H)⁴⁺Calculated (M)=4472

Example 32N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)

Preparation analogous to SPPS Method B.HPLC method 02_B6_(—)1:RT=33.58 minLCMS: m/z=1114 (M+4H)⁴⁺Calculated (M)=4451.1

Example 33N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxy-heptadecanoylamino)-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl]-[Aib8,Glu22,Arg26,Arg34, Aib35, Lys37]GLP-1-(7-37)

Preparation analogous to SPPS Method B.UPLC method 04_A3_(—)1:RT=9.81 minLCMS: m/z=1079 (M+4H)⁴⁺Calculated (M)=4312.9

Example 34N-epsilon31-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Glu22,Val25,Arg26,Lys31,Arg34,Arg35,Arg37]GLP-1-(7-37)

Preparation method: The peptide was prepared by SPPS Method C and thefinal product was characterized by analytical HPLC and MALDI-MS.

HPLC (02-B4-4): RT=7.9 min

HPLC (neutral, 30-60% over 16 min): RT=5.6 min

MALDI-MS: 4395

Calculated MW=4397

Example 35N-epsilon31{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-[Aib8,Glu22,Val25,Arg26,Lys31,Arg34,Arg35,Arg37)]GLP-1(7-37)yl[Arg39,Arg40,Arg41]

Preparation method: The peptide was prepared by SPPS Method C and thefinal product was characterized by analytical HPLC and LC-MS.

HPLC (02-B4-4): RT=7.36 min

HPLC (neutral): RT=17.5 min

MALDI-MS: 5022

Calculated MW=5021.8

Example 36N-epsilon31-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Glu22,Val25,Arg26,Lys31,Lys35,Lys36]GLP-1-(7-36)amide

Preparation method: The peptide was prepared by SPPS Method C and thefinal product was characterized by analytical HPLC and MALDI-MS.

HPLC (02-B4-2): RT=8.4 min

HPLC (04-A3-1): RT=7.8 min

MALDI-MS: 4141

Calculated MW=4140.8

Example 37

N-epsilon31-{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-N-beta34-(2-(bis-carboxymethylamino)acetyl)[Aib8,Glu22,Val25,Arg26,Lys31,Dap34] GLP-1(7-34) amide

Preparation method: The peptide was prepared by SPPS Method C and thefinal product was characterized by analytical HPLC and MALDI-MS.

HPLC (02-B6-1): RT=36.7 min

HPLC (A4-A3-1): RT=8.9 min

MALDI-MS: 4015.9

Calculated MW=4015.5

Example 38N-epsilon-37-[(S)-4-carboxy-4-(2-{2-[2-(2-{2-[2-(17-carboxyheptadecanoylamino)ethoxy]ethoxy}acetylamino) ethoxy]ethoxy}acetylamino)butyryl][Aib8,Glu22,Arg26,34,Lys37] GLP-1 (7-37)

Preparation analogous to SPPS Method B.HPLC method 04_A3_(—)1:RT=10.26 minLCMS: m/z=1071.7 (M+4H)⁴⁺Calculated (M)=4284.9Biological Findings

Protraction of GLP-1 Derivatives after i.v. or s.c. Administration

The protraction of a number GLP-1 derivatives of the invention may bedetermined by monitoring the concentration thereof in plasma after scadministration to healthy pigs, using the methods described below. Forcomparison also the concentration in plasma of GLP-1(7-37) after sc.administration may be followed. The protraction of other GLP-1derivatives of the invention can be determined in the same way.

Example 39 Pharmacokinetic Testing in Minipigs

A number of GLP-1 derivatives of the invention (the compounds ofExamples 1, 4, 5, 7, 8, 9, 13, and 22) were subjected to pharmacokinetictesting in minipigs. Liraglutide was included in the test forcomparative purposes. A second comparative compound was also included,viz. a GLP-1 derivative with the substitutions (22E+26R), which isdescribed in Example 63 of WO 2005/027978.

Generally, the test substances are to be dissolved in a vehicle suitablefor subcutaneous or intravenous administration. The concentration is tobe adjusted so the dosing volume is approximately 1 ml. In the presentstudy the test substances were administered subcutaneously.

The study was performed in 12 male Göttingen minipigs from EllegaardGöttingen Minipigs ApS. An acclimatisation period of approximately 10days was allowed before the animals entered the study. At start of theacclimatisation period the minipigs were about 5 months old and in theweight range of 8-10 kg.

The study was conducted in a suitable animal room with a roomtemperature set at 21-23° C. and the relative humidity to 50%. The roomwas illuminated to give a cycle of 12 hours light and 12 hours darkness.Light was from 06.00 to 18.00 h. The animals were housed in pens withstraw as bedding, six together in each pen. The animals had free accessto domestic quality drinking water during the study, but were fastedfrom approximately 4 μm the day before dosing until approximately 12hours after dosing. The animals were weighed on arrival and on the daysof dosing.

The animals received a single subcutaneous injection. The subcutaneousinjection was given on the right side of the neck, approximately 5-7 cmfrom the ear and 7-9 cm from the middle of the neck. The injections weregiven with a stopper on the needle, allowing 0.5 cm of the needle to beintroduced. Each test substance was given to three animals. Each animalreceived a dose of 2 nmol/kg body weight. Six animals were dosed perweek while the remaining six were rested.

A full plasma concentration-time profile was obtained from each animal.Blood samples were collected according to the following schedule:

After Intravenous Administration (not Applicable in the Present Study):

Predose (O), 0.17 (10 minutes), 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72, 96,and 120 hours after injection.

After Subcutaneous Administration:

Predose (O), 0.5, 1, 2, 4, 6, 8, 12, 24, 48, 72, 96, and 120 hours afterinjection.

At each sampling time, 2 ml of blood was drawn from each animal. Theblood samples were taken from a jugular vein.

The blood samples were collected into test tubes containing a buffer forstabilisation in order to prevent enzymatic degradation of the GLP-1derivatives.

Plasma was immediately transferred to Micronic-tubes. Approximately 200μl plasma was transferred to each Micronic-tube. The plasma was storedat −20° C. until assayed. The plasma samples were assayed for thecontent of GLP-1 derivatives using an immunoassay.

The plasma concentration-time profiles were analysed by anon-compartmental pharmacokinetic analysis. The followingpharmacokinetic parameters were calculated at each occasion: AUC,AUC/Dose, AUC_(%Extrap), C_(max), t_(max), λ_(z), t_(1/2), CL, CL/f,V_(z), V_(z)/f and MRT.

All tested GLP-1 derivatives of the invention had a half-life aftersubcutaneous administration in minipigs in the range of 40-100 hours.The half-lives of the comparative compounds liraglutide and Compound 135were 18 hours, and 28 hours, respectively.

Selected derivatives of the invention are tested in Danish Landracepigs.

Pharmacokinetic Testing of GLP-1 Derivatives in Pigs

Pigs (50% Duroc, 25% Yorkshire, 25% Danish Landrace, app 40 kg) arefasted from the beginning of the experiment. To each pig 0.5 nmol oftest derivative per kg body weight is administered in a 50 μM isotonicsolution (5 mM phosphate, pH 7.4, 0.02% Tween®-20 (Merck), 45 mg/mlmannitol (pyrogen free, Novo Nordisk). Blood samples are drawn from acatheter in vena jugularis. 5 ml of the blood samples are poured intochilled glasses containing 175 μl of the following solution: 0.18 MEDTA, 15000 KIE/ml aprotinin (Novo Nordisk) and 0.30 mMValine-Pyrrolidide (Novo Nordisk), pH 7.4. Within 30 min, the samplesare centrifuged for 10 min at 5-6000*g. Temperature is kept at 4° C. Thesupernatant is pipetted into different glasses and kept at minus 20° C.until use.

The plasma concentrations of the peptides are determined in a sandwichELISA or by RIA using different mono- or polyclonal antibodies. Choiceof antibodies depends of the GLP-1 derivatives. The time at which thepeak concentration in plasma is achieved varies within wide limits,depending on the particular GLP-1 derivative selected.

General Assay Protocol for Sandwich ELISA in 96-Wells Microtiterplate

Coating buffer (PBS): Phosphate buffered saline, pH 7.2 Wash-buffer(PBS-wash): Phosphate buffered saline, 0.05% v/v Tween 20, pH 7.2Assay-buffer (BSA-buffer): Phosphate buffered saline, 10 g/l Bovin SerumAlbumin (Fluka 05477), 0.05% v/v Tween 20, pH 7.2 Streptavidin-bufferPhosphate buffered saline, 0.5M NaCl, 0.05% v/v Tween 20, pH 7.2Standard: Individual derivatives in a plasma-matrix A-TNP: Nonsensantibody AMDEX: Streptavin-horseradish-peroxodase (Amersham RPN4401V)TMB-substrate: 3,3′,5,5′tetramethylbenzidine (<0.02%), hydrogen peroxide

The assay was carried out as follows (volumen/well):

-   1.) coat with 100 μl catching antibody 5 μg/ml in PBS-buffer    -   incubate o/n, 4° C.    -   5×PBS-wash    -   blocked with last wash in minimum 30 minute    -   then empty the plate-   2.) 20 μl sample+100 μl biotinylated detecting antibody 1 μg/ml in    BSA-buffer with 10 μg/ml A-TNP    -   incubate 2 h, room temperature, on a shaker □ 5×PBS-wash, then        empty the plate-   3.) 100 μl AMDEX 1:8000 in Streptavidin-buffer    -   incubate 45-60 minute, room temperature, on a shaker    -   5×PBS-wash, then empty the plate-   4.) 100 μl TMB-substrate    -   incubate x minute at room temperature on a shaker    -   stop the reaction with 100 μl 4 M H₃PO₄

Read the absorbance at 450 nm with 620 nm as reference

The concentration in the samples was calculated from standard curves.

General Assay Protocol for RIA

DB-buffer: 80 mM phosphate buffer, 0.1% Human serum albumin, 10 mM EDTA,0.6 mM thiomersal, pH 7.5 FAM-buffer: 40 mM phosphate buffer, 0.1% HumanSerum Albumin, 0.6 mM thiomersal, pH 7.5 Charcoal: 40 mM phosphatebuffer, 0.6 mM thiomersal, 16.7% bovine plasma, 15 g/l activated carbon,pH 7.5 (mix the suspension minimum 1 h before use at 4° C.) Standard:Individual derivatives in a plasma-matrix

The assay was carried out in minisorp tubes 12×75 mm (volumen/tube) asfollows:

Db-buffer SAMPLE Antibody FAM-buf. Tracer Charcoal H₂O Day 1 Total 100μL NSB 330 μL 100 μL Sample 300 μL 30 μL 100 μL 100 μL Mix, incubate o/nat 4° C. Day 2 Total 1.5 mL NSB 1.5 mL Sample 1.5 mL

Mix—incubate 30 min at 4° C.—centrifuge at 3000 rpm, 30 min—immediatelyafter transfer supernatants to new tubes, close with stopper and counton gamma-counter for 1 minute.

The concentration in the samples was calculated from individual standardcurves.

GLP-1 Radio Receptor Assay (RRA):

The method is a radiometric-ligand binding assay using LEADseekerimaging particles. The assay is composed of membrane fragmentscontaining the GLP-1 receptor, unlabeled GLP-1 analogues, human GLP-1labelled with ¹²⁵I and PS LEADseeker particles coated with wheat germagglutinin (WGA). Cold and ¹²⁵I-labelled GLP-1 will compete for thebinding to the receptor. When the LEADseeker particles are added theywill bind to carbohydrates residues on the membrane fragments via theWGA-residues. The proximity between the ¹²⁵I-molecules and theLEADseeker particles causes light emission from the particles. TheLEADseeker will image the emitted light and it will be reversiblycorrelated to the amount of GLP-1 analogue present in the sample.

Reagents & Materials:

Pre treatment of animal plasma: Animal plasma was heat treated for 4 hrsat 56° C. and centrifuged at 10.000 rpm for 10 minutes. Afterwards,Val-Pyr (10 μM) and aprotenin (500 KIE/mL) was added and stored at <−18°C. until use.

GLP-1 analogues calibrators: GLP-1 analogues were spiked intoheat-treated plasma to produce dilution lines ranging from approximately1 μM to 1 μM.

GLP-1 RRA assay buffer: 25 mM Na-HEPES (pH=7.5), 2.5 mM CaCl₂, 1 mMMgCl₂, 50 mM NaCl, 0.1% ovalbumin, 0.003% tween 20, 0.005% bacitracin,0.05% NaN₃.

GLP-1 receptor suspension: GLP-1 receptor membrane fragments werepurified from baby hamster kidney (BHK) cells stably expressing thehuman pancreatic GLP-1 receptor. Stored <−80° C. until use.

WGA-coupled polystyrene LEADseeker imaging beads (RPNQ0260, Amersham):The beads were reconstituted with GLP-1 RRA assay buffer to aconcentration of 13.3 mg/mL. The GLP-1 receptor membrane suspension wasthen added and incubated cold (2-8° C.) at end-over-end for at least 1hr prior to use.

[¹²⁵I]-GLP-1(7-36)amide (Novo Nordisk A/S). Stored <−18° C. until use.

Ethanol 99.9% vol (De Dansk Spritfabrikker A/S): Stored <−18° C. untiluse.

MultiScreen® Solvinert 0.45 μm hydrophobic PTFE plates (MSRPN0450,Millipore Corp.)

Poly propylene plates (cat. no. 650201, Greiner Bio-One)

White polystyrene 384-well plates (cat. no. 781075, Greiner Bio-One)

Apparatus:

Horizontal Plate Mixer

Centrifuge with a standard swinging-bucket microtitre plate rotorassembly

UltraVap—Drydown Sample Concentrator (Porvair)

LEADseeker™ Multimodality Imaging System (Amersham)

Assay Procedure:

Sample Preparation:

Mount the MultiScreen® Solvinert filter plate on a chemical-comparablereceiver plate (i.e. poly propylene plates) to collect the filtrate.

Add 150 μL ice-cold ethanol 99.9% into the empty wells of theMultiScreen® Solvinert filter plate followed by 50 μL calibrator orplasma sample. Place the storage lid on the filter plate.

Incubate 15 minutes at 18-22° C. on a horizontal plate mixer.

Place the assembled filter and receiver plate, with the lid, into astandard swinging-bucket microtitre plate rotor assembly. The filtrateis then collected in the empty wells of the receiver plate at 1500 rpmfor 2 minutes.

Dry down the filtrate by using the UltraVap with heated (40° C.) N₂ forduration of 15 minutes. Reconstitute the dry material by adding 100 μLGLP-1 RRA assay buffer into each well. Incubate for 5 minutes on ahorizontal mixer.

GLP-1 Radio Receptor Assay:

Use the following pipetting scheme and white polystyrene 384-wellplates:

-   -   35 μL GLP-1 RRA assay buffer    -   5 μL reconstituted filtrate.    -   10 μL [¹²⁵I]-GLP-1(7-36)amide. The stock solution was diluted in        GLP-1 RRA assay buffer to 20.000 cpm/well prior to use.    -   15 μL GLP-1 receptor membrane fragments (≈0.5 μg/well)        pre-coated to WGA-polystyrene LEADseeker imaging beads (0.2        mg/well)

Seal the plates and incubate over night at 18-22° C.

The light emission from each wells are detected by using the LEADseeker™Multimodality Imaging System for duration of 10 minutes.

Example 40 Stimulation of cAMP Formation in a Cell Line Expressing theCloned Human GLP-1 Receptor

The potencies of a number of GLP-1 derivatives of the invention weredetermined as described below, i.e. as the stimulation of the formationof cyclic AMP (cAMP) in a medium containing the human GLP-1 receptor.For comparison, the potencies of two known GLP-1 derivates were alsodetermined, viz. that of liraglutide, and that of Compound 135, a GLP-1derivative with the substitutions (22E+26R), which is described inExample 63 of WO 2005/027978.

Purified plasma membranes from a stable transfected cell line,BHK467-12A (tk-ts13), expressing the human GLP-1 receptor was stimulatedwith the GLP-1 derivative in question, and the potency of cAMPproduction was measured using the AlphaScreen™ cAMP Assay Kit fromPerkin Elmer Life Sciences.

A stable transfected cell line has been prepared at NN A/S, Denmark, anda high expressing clone was selected for screening. The cells were grownat 5% CO₂ in DMEM, 5% FCS, 1% Pen/Strep (Penicillin/Streptomycin) and0.5 mg/ml of the selection marker G418.

Cells at approximate 80% confluence were washed 2× with PBS (PhosphateBuffered Saline) and harvested with Versene (aqueous solution of thetetrasodium salt of ethylenediaminetetraacetic acid), centrifuged 5 minat 1000 rpm and the supernatant removed. The additional steps were allmade on ice. The cell pellet was homogenized by the Ultrathurax for20-30 sec. in 10 ml of Buffer 1 (20 mM Na-HEPES, 10 mM EDTA, pH=7.4),centrifuged 15 min at 20.000 rpm and the pellet resuspended in 10 ml ofBuffer 2 (20 mM Na-HEPES, 0.1 mM EDTA, pH=7.4). The suspension washomogenized for 20-30 sec and centrifuged 15 min at 20.000 rpm.Suspension in Buffer 2, homogenization and centrifugation was repeatedonce and the membranes were resuspended in Buffer 2 and ready forfurther analysis or stored at −80° C.

The functional receptor assay was carried out by measuring the peptideinduced cAMP production by The AlphaScreen Technology. The basicprinciple of The AlphaScreen Technology is a competition betweenendogenous cAMP and exogenously added biotin-cAMP. The capture of cAMPis achieved by using a specific antibody conjugated to acceptor beads.Formed cAMP was counted and measured at a AlphaFusion MicroplateAnalyzer. The EC₅₀ values were calculated using the Graph-Pad Prismesoftware (version 5).

TABLE 1 Potency of GLP-1 derivatives Compound of Example No. Potency[EC₅₀/nM] 1 0.37 2 0.20 3 0.35 4 0.52 5 0.20 6 0.18 7 0.58 8 0.66 9 0.5010 0.66 11 0.61 12 1.80 13 1.97 14 0.29 15 0.03 16 0.15 17 0.16 18 0.0219 0.05 20 0.22 21 1.27 22 1.35 23 0.02 24 0.01 25 0.36 26 0.25 27 0.0528 0.08 29 0.10 30 0.21 31 0.04 32 0.46 33 0.06 34 1.27 35 0.74 36 0.1437 0.34 38 0.07 Liraglutide 0.10 Compound 135 4.00

As it is apparent from Table 1, very many of the GLP-1 derivatives aremore potent than the prior art GLP-1 derivative of liraglutide (thelower the EC₅₀ value the better). All GLP-1 derivatives of the inventionare much more potent than the Compound 135.

Example 41 Albumin Binding Affinity

The affinities of a number of GLP-1 derivatives of the invention forhuman serum albumin (HSA) were measured by a competition scintillationproximity assay (SPA) as described in the following.

Streptavidin-SPA beads (GE Healthcare RPNQ0009) were incubated withbiotinylated HSA for 5 hours. The beads were washed with buffer toremove unbound HSA. The beads were mixed with a ¹²⁵I-labeled acylatedGLP-1 analogue(N-epsilon37-[2-(2-[2-((S)-4-((S)-4-(12-[4-(16-(1H-tetrazol-5-yl)hexadecanoylsulfamoyl)butyrylamino]dodecanoylamino)-4-carboxybutyrylamino)-4-carboxybutyrylamino)ethoxy]ethoxy)acetyl][Aib8,¹²⁵I-Tyr19,Glu22,Arg26,Arg34,Lys37]GLP-1(7-37)-NH₂)in a buffer containing 100 mM Hepes, 100 mM NaCl, 10 mM MgSO₄, 0.025%Tween-20, pH 7.4. The mixture was pipetted into the wells of a PerkinElmer Optiplate-96 6005290 (100 μl per well) and 100 μl of a dilutionseries of the GLP-1 derivative to be measured was added in the samebuffer. After 20 hours of gentle rocking at room temperature the plateswere centrifuged and counted on a TopCounter. Bound cpm was plotted as afunction of GLP-1 derivative concentration and the EC₅₀ value of thecompetition curve was used as a measure of the affinity of thederivative for HSA.

The albumin binding affinities (EC₅₀, in nM) of various GLP-1derivatives of the invention are shown in Table 2 below.

TABLE 2 Albumin binding affinity Compound of Example No. Albumin bindingaffinity (EC₅₀/nM) 11, 26, 32  1-100 5, 13, 27, 33 100-150 14, 25, 37150-200 2, 4, 6, 20, 28 200-400 1, 22, 29, 36 400-800 7, 9, 12, 35 800-1500 3, 8, 23, 24, 31 1500-2000 18, 21, 30, 34 above 2000

As it is apparent from Table 2, several of the GLP-1 derivatives of theinvention have a high albumin binding affinity corresponding to an EC₅₀of below 2000 nM (the lower the EC₅₀, the higher the albumin bindingaffinity).

Example 42 Binding to the Extracellular Domain of the GLP-1 Receptor

For a number of GLP-1 derivatives of the invention (the compounds ofExamples 1, 3, 4, 5, 6, 14, 15, 16, 17, 18, 19, 21, 22, 23, and 24), theaffinity of the binding to the isolated N-terminal extracellular domainof the GLP-1R receptor (nGLP-1R) was measured as described below.Liraglutide was included for comparison.

The affinity is a measure of the ability of the GLP-1 derivative inquestion to displace ¹²⁵I-Exendin-4(9-39) from binding to nGLP-1R, andthe binding to nGLP-1R was measured in the following assay: The proteinnGLP-1R was prepared as described by Runge et al 2007 (Biochemistry,vol. 46, pp. 5830-5840), biotinylated and immobilized onstreptavidin-coated SPA beads. The nGLP1R in 0.1M NaHCO₃ wasbiotinylated using 75 μg BNHS (Sigma H1759) to 1 mg protein. Thebiotinylated nGLP1R was subsequently dialyzed against PBS. All reagentsand derivatives were diluted in PBS with 0.05% v/v Tween 20. The bindingassay was carried out in 96 well OptiPlates (PerkinElmer 6005290) in afinal volume of 200 μl. Each well contained 2 mg streptavidin coated SPAbeads (PerkinElmer RPNQ007), 0.1 pmol biotinylated nGLP1R, 50 pCi¹²⁵I-Exendin (9-39) and test peptide in final concentrations rangingfrom 1000 nM to 0.064 nM. The plates were incubated on a shaker at RTfor 3 hours. The SPA particles were spun down by centrifugation for 10min at 1500 rpm and the plates were counted in a TopCount-NXT(PerkinElmer).

The IC₅₀ value is read from the respective curve as the concentration ofthe GLP-1 derivative in question which displaces 50% of¹²⁵I-Exendin-4(9-39) from binding to nGLP-1R.

The affinity of liraglutide to nGLP-1R (IC₅₀) was determined to 1500 nM.The tested GLP-1 derivatives of the invention had affinities rangingfrom 2.0-276 (nM), with six compounds in the range of 2.0-10.0 nM. Alltested GLP-1 derivatives of the invention accordingly exhibit animproved binding affinity to the N-terminal GLP-1 receptor, relative toliraglutide (the lower the IC₅₀ value, the better the binding).

Example 43 Dose-Response Study in db/db Mice

A number of GLP-1 derivatives of the invention (the compounds ofExamples 1, 7, 8, 9, 11, 12, 13, 20, 22, and 27) were tested in adose-response study in an obese, diabetic mouse model (db/db mice) asdescribed in the following.

Fifty db/db mice (Taconic, Denmark), 10-12 weeks of age, were housedaccording to standard animal welfare rules of Novo Nordisk and weregiven free access to standard chow (e.g. Altromin 1324, Brogaarden,Gentofte, Denmark) and tap water and kept at 24° C. After 1 week ofacclimatisation, the basal blood glucose was assessed twice. Based onthe mean blood glucose values, 42 mice were selected for furtherexperimentation and allocated to 7 groups (n=6) with matching bloodglucose levels. The mice were used in experiments of 3-6 days' durationfor up to 4 times, following which they were euthanized.

The seven groups received treatment as follows:

1: Vehicle, s.c.

2: GLP-1 derivative, 0.3 nmol/kg, s.c.

3: GLP-1 derivative, 1 nmol/kg, s.c.

4: GLP-1 derivative, 3 nmol/kg, s.c.

5: GLP-1 derivative, 10 nmol/kg, s.c.

6: GLP-1 derivative, 30 nmol/kg, s.c.

7: GLP-1 derivative, 100 nmol/kg, s.c.

Vehicle: 50 mM phosphate, 0.05% tween 80, pH 8. The GLP-1 derivative wasdissolved in the vehicle, e.g. to concentrations of 0.05, 0.17, 0.5,1.7, 5 and 17 nmol/ml and 300 microliter were administered s.c. permouse weighing 50 g (6 ml/kg).

On the day of dosing, the compound in question was dosed atapproximately 9 am (time 0). At time −½ h (8.30 am) blood glucose wasassessed, following which the mice were weighed. Blood glucose wasassessed several times on the day of dosing, usually at time 1, 3 and 6h (10 am, 12 am and 3 pm).

On the following days, the blood glucose was assessed at time 24, 48,72, and 96 h after dosing (i.e. at 9 am on day 2, 3, 4, 5), followed byweighing. In some studies, blood glucose and body weight was furthermoreassessed 120 h (day 6) after dosing.

The mice were weighed individually on a digital weight.

Samples for the measurement of blood glucose were obtained from the tailtip capillary of conscious mice. Blood, 10 μl, was collected intoheparinised capillaries and transferred to 500 μl glucose buffer (EBIObuffer solution, Eppendorf, Germany). The glucose concentration wasmeasured using the glucose oxidase method (glucose analyser Biosen 5040,EKF Diagnostic, GmbH, Barleben, Germany). The samples were kept at roomtemperature for up to 1 h until analysis. If analysis had to bepostponed, samples were kept at 4° C. for a maximum of 24 h.

The half-lives (T½) were calculated according to the followingmathematical model: Assuming that

(1) the disappearance of the compounds from plasma is monoexponential;

(2) the effect on blood glucose (deltaBG) can be described by a standardsigmoidal dose-response curve;

(3) the first 6 hours of absorption and distribution are ignored andonly the return of the glucose from the bottom to the baseline (minimumto 0) is fitted; then the glucose response (Y) (for example deltaBG) canbe described by the following equation:Y=Bottom+(Top−Bottom)/(1+Dose*exp(−ln 2*t/T1/2)/ED50),where the variables ED50 and T½ are defined as follows:ED50 is the dose giving rise to half maximal effect on BG (in nmol/kg)T1/2 is the half-life (in hours); and the following are globalConstants:Top (the response after return to baseline glucose), and Bottom (theresponse at maximal glucose fall); and the following is a constant foreach data set (each dose):Dose (the administered dose (in nmol/kg)).

All data sets are fitted simultaneously.

All compounds tested had a half-life (T½) in the range of 10-30 hours.

Example 44 Alpha-Helix Content

The content of α-helical (alpha-helical) structure for a number of GLP-1derivatives of the invention (the compounds of Examples 3, 4, 5, 7, 8,9, 11, 12, 13, 14, 21, 22, and 24) was estimated using circulardichroism (CD) spectroscopy, as described below. Liraglutide wasincluded in the test for comparative purposes.

Far-UV circular dichroism spectra were recorded on a Jasco J-715spectropolarimeter on 5 μM (uM, micro-Molar) peptide in 10 mM Tris/ClO₄pH 8.0. Raw data were subtracted buffer background and normalised tomolar ellipticity in units of M⁻¹ cm⁻¹ based on the concentration ofpeptide bonds and the intensity value at 222 nm was extracted.

The intensity at 222 nm was used as a measure of alpha-helical content,i.e the signal is proportional to the alpha-helical content such that avalue of −1 M⁻¹ cm⁻¹ corresponds to 10% of alpha-helical structure(Venyaminov and Yang 1996: “Determination of protein secondarystructure” In Circular Dichroism and conformational analysis ofbiomolecules (Ed. Fasman G D, Plenum Press NY), pp. 69-108).

From the spectra, the values for the molar ellipticity (Δε, or deltaepsilon) were derived and the corresponding alpha-helical contentestimated. The overall spectra features of the GLP-1 derivatives in therange of 200-250 nm are described by two bands with minima at 206 nm and222 nm respectively. This is in accordance with a secondary structurewith a significant alpha-helical stretch.

The value of delta epsilon at 222 nm for liraglutide in 5 uMconcentration was below −3.6, corresponding to an alpha-helical contentof liraglutide of below 36%.

The values of delta epsilon at 222 nm for the compounds of Examples 4,5, 11, and 14 in 5 uM concentration were in the range of −2.0 to −3.6,corresponding to alpha-helical contents in the range of 20-36%.

The values of delta epsilon at 222 nm for the compounds of Examples 3,7, 8, 9, 12, 13, 21, 22, and 24 in 5 uM concentration were in the rangeof −3.7 to −6.0, corresponding to alpha-helical contents in the range of37-60%.

The invention claimed is:
 1. A GLP-1 derivative which comprises amodified GLP-1(7-37) sequence having: i) a total of 3-12 amino acidmodifications as compared to GLP-1(7-37) (SEQ ID No: 1), including a) aAib residue at a position equivalent to position 8 of GLP-1(7-37), b) aGlu residue at a position equivalent to position 22 of GLP-1(7-37), andc) an Arg residue at a position equivalent to position 26 ofGLP-1(7-37); and ii) which is derivatised with an albumin bindingresidue or pegylated in a position selected from a position equivalentto position 18, 20, 23, 30, 31, 34, 36, or 37 of GLP-1(7-37).
 2. TheGLP-1 derivative according to claim 1, which comprises 3-8 amino acidsubstitutions as compared to GLP-1(7-37) (SEQ ID No: 1).
 3. The GLP-1derivative according to claim 1, which comprises 1-3 amino aciddeletions as compared to GLP-1(7-37) (SEQ ID No: 1).
 4. The GLP-1derivative according to claim 1 which comprises 1-4 amino acid additionsas compared to GLP-1(7-37) (SEQ ID No: 1).
 5. The GLP-1 derivativeaccording to claim 1, which has a free C-terminal carboxylic acid group.6. The GLP-1 derivative according to claim 1 having a sequence accordingto formula (I): Formula (I) (SEQ ID No: 2)Xaa₇-Xaa₈-Xaa₉-Gly-Thr-Phe-Thr-Ser-Asp-Xaa₁₆-Ser-Xaa₁₈-Tyr-Xaa₂₀-Glu-Glu-Xaa₂₃-Xaaz4-Xaa₂₄-Arg-Xaa₂₇-Xaa₂₈-Ile-Xaa₃₀-Xaa₃₁-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇-Xaa₃₈-Xaa₃₉-Xaa₄₀-Xaa₄₁-R

wherein (Xaa₇-Xaa₈) is (L-histidine-Aib), (desamino-histidine-Aib), or(desamino-histidine-Aib); Xaa₉ is Glu, or a Glu derivative; Xaa₁₆ isVal, or Leu; Xaa₁₈ is Ser, Lys, Cys, or Arg; Xaa₂₀ is Leu, or Lys; Xaa₂₃is Gln, Glu, Lys, Cys, or Arg; Xaa₂₄ is Ala, or Asn; Xaa₂₅ is Ala, orVal; Xaa₂₇ is Glu, Ala, or Leu; Xaa₂₈ is Phe, or a Phe derivative; Xaa₃₀is Ala, Glu, Lys, or Arg; Xaa₃₁ is Trp, Cys, or Lys; Xaa₃₃ is Val, Cys,or Lys; Xaa₃₄ is Lys, Cys, Glu, Asn, Dap, or Arg; Xaa₃₅ is Gly, Arg,Lys, Aib, or absent; Xaa₃₆ is Arg, Lys, or absent; Xaa₃₇ is Gly, Aib,Cys, Lys, epsilon-amino-Lys, Pro, Arg, or absent; Xaa₃₈ is Lys, Glu,Arg, or absent; Xaa₃₉ is Lys, Arg, or absent; Xaa₄₀ is Arg, or absent;Xaa₄₁ is Arg, or absent; and R is amide, or absent; provided that ifXaa₃₇, Xaa₃₈, Xaa₃₉, or Xaa₄₀ is absent, then each amino acid residuedownstream is also absent; and which is derivatised with an albuminbinding residue or pegylated in a position selected from a positionequivalent to position 18, 20, 23, 30, 31, 34, 36, 37, or 39 of GLP-1(7-37) (SEQ ID No: 1).
 7. The GLP-1 derivative according to claim 1having a sequence according to formula (II): Formula (II) (SEQ ID No: 3)Xaa₇-Xaa₈-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Xaa₁₈-Tyr-Leu-Glu-Glu-Gln-Ala-Ala-Arg-Glu-Phe-Ile-Xaa₃₀-Xaa₃₁-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-Xaa₃₇- Xaa₃₈-Xaa₃₉-Xaa₄₀-Xaa₄₁-R

wherein Xaa₇ is L-histidine, D-histidine, desamino-histidine,2-amino-histidine, β-hydroxy-histidine, homohistidine,N^(α)-acetyl-histidine, α-fluoromethyl-histidine, α-methyl-histidine,3-pyridylalanine, 2-pyridylalanine, or 4-pyridylalanine; Xaa₈ is Aib;Xaa₁₈ is Ser, Lys, or Arg; Xaa₃₀ is Ala, Glu, Lys, or Arg; Xaa₃₁ is Lys,or Trp; Xaa₃₃ is Val; Xaa₃₄ is Lys, Glu, Dap, or Arg; Xaa₃₅ is Gly, Arg,Aib, or absent; Xaa₃₆ is Arg, Lys, or absent; Xaa₃₇ is Gly, Aib, Lys,epsilon-amino-Lys, Pro, Arg, or absent; Xaa₃₈ is Glu, Arg, or absent;Xaa₃₉ is Arg, or absent; Xaa₄₀ is Arg, or absent; Xaa₄₁ is Arg, orabsent; and R is amide, or is absent; provided that if Xaa₃₇, Xaa₃₈,Xaa₃₉, or Xaa₄₀ is absent, then each amino acid residue downstream isalso absent; and which is derivatised with an albumin binding residue orpegylated in a position selected from a position equivalent to position18, 23, 30, 31, 34, 36 or 37 of GLP-1(7-37) (SEQ ID No: 1).
 8. TheGLP-derivative according to claim 1 which is selected from thefollowing:N-epsilon37{2-[2-(2-{2-[2-((S)-3-carboxy-3-{[1-(19-carboxynonadecanoyl)piperidine-4-carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl[Aib8,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide;N-epsilon30{2-[2-(2-{2-[2-((S)-3-carboxy-3-{[1-(19-carboxynonadecanoyl)piperidine-4-carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl[Aib8,Glu22,Arg26,Lys30]GLP-1-(7-37);N-epsilon31{2-[2-(2-{2-[2-((S)-3-carboxy-3-{[1-(19-carboxynonadecanoyl)piperidine-4-carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl[Aib8,Glu22,Arg26,Lys31]GLP-1-(7-37);N-epsilon31-(2-{2-2[2-{2-[2-((S)-3-Carboxy-3-{[1-(19-carboxy-nonadecanoyl)piperidine-4-carbonyl]amino}propionylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetyl)[Aib8,Glu22,Arg26,Lys31,Arg34]GLP-1-(7-37);N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxy-nonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide;N-epsilon20-[2-(2-{2-[(S)-4-Carboxy-4-((S)-4-carboxy-4-{12-[4-(16-(1H-tetrazol-5-yl)hexadecanoylsulfamoyl)butyrylamino]dodecanoylamino}butyrylamino)butyrylamino]ethoxy}ethoxy)acetyl][Aib8,Lys20,Glu22,Arg26,Glu30,Pro37]GLP-1-(7-37)amide;N-epsilon37-[2-(2-{2-[(S)-4-Carboxy-4-((S)-4-carboxy-4-{12-[4-(16-(1H-tetrazol-5-yl)hexadecanoylsulfamoyl)butyrylamino]dodecanoylamino}butyrylamino)butyrylamino]ethoxy}ethoxy)acetyl][Aib8,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37)amide;[Aib8,Glu22,Arg26,Glu30,Pro37]GLP-1-(7-37)Lys[2-(2-{2-[4-Carboxy-4-(4-carboxy-4-{4-[4-(16-1H-tetrazol-5-yl-hexadecanoylsulfamoyl)butyrylamino]butyrylamino}butyrylamino)butyrylamino]ethoxy}ethoxy)acetyl];N-epsilon36-(2-(2-(2-((2-[2-(2-(17-carboxyheptadecanoylamino)ethoxy)ethoxy]acetylamino)ethoxy)ethoxy)acetyl)[Aib8,Glu22,Arg26,Glu30,Lys36]GLP-1-(7-37)Glu-amide;N-epsilon31-[2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamino]ethoxy]ethoxy}acetylamino]ethoxy}ethoxy)acetyl][Aib8,Glu22,Arg26,Lys31]GLP-1-(7-37);N-epsilon20-(2-{2[2-(2-{2-[2-((S)-4-Carboxy-4-hexadecanoylamino-butyrylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}-acetyl)[Aib8,Lys20,Glu22,Arg26,Glu30,Pro37]GLP-1-(7-37)amide;N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxy-heptadecanoylamino)-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl][Aib8,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37);N-alpha37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxy-heptadecanoylamino)-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl][Aib8,Glu22,Arg26,Arg34,epsilon-Lys37]GLP-1-(7-37)peptide;N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-({trans-4-[(19-carboxynonadecanoylamino)methyl]cyclohexanecarbonyl}amino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Glu22,Arg26,Arg34,Lys37]GLP-1-(7-37);N-epsilon37-[2-(2-{2-[2-(2-{2-[(S)-4-carboxy-4-(17-carboxy-heptadecanoylamino)-butyrylamino]-ethoxy}-ethoxy)-acetylamino]-ethoxy}-ethoxy)-acetyl]-[Aib8,Glu22,Arg26,Arg34,Aib35,Lys37]GLP-1-(7-37);N-epsilon31-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Glu22,Val25,Arg26,Lys31,Arg34,Arg35,Arg37]GLP-1-(7-37);N-epsilon31{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-[Aib8,Glu22,Val25,Arg26,Lys31,Arg34,Arg35,Arg37)]GLP-1(7-37)yl[Arg39,Arg40,Arg41];N-epsilon31-[2-(2-{2-[2-(2-{2-[(S)-4-Carboxy-4-(17-carboxyheptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl][Aib8,Glu22,Val25,Arg26,Lys31,Lys35,Lys36]GLP-1-(7-36) amide;N-epsilon31-{2-(2-{2-[2-(2-{2-[4-Carboxy-4-(17-carboxy-heptadecanoylamino)butyrylamino]ethoxy}ethoxy)acetylamino]ethoxy}ethoxy)acetyl}-N-beta34-(2-(bis-carboxymethylamino)acetyl)[Aib8,Glu22,Val25,Arg26,Lys31,Dap34]GLP-1(7-34) amide; andN-epsilon-37-[(S)-4-carboxy-4-(2-{2-[2-(2-{2-[2-(17-carboxyheptadecanoylamino)ethoxy]ethoxy}acetylamino)ethoxy]ethoxy}acetylamino) butyryl][Aib8,Glu22,Arg26,34,Lys37]GLP-1(7-37).
 9. A pharmaceutical composition comprising a derivativeaccording to claim 1 or a pharmaceutically acceptable salt, amide,alkyl, or ester thereof, and a pharmaceutically acceptable excipient.10. A method of treating hyperglycemia, type 2 diabetes, impairedglucose tolerance, type 1 diabetes, obesity, hypertension, syndrome X,dyslipidemia, cognitive disorders, atheroschlerosis, myocardialinfarction, coronary heart disease, stroke, inflammatory bowel syndrome,dyspepsia and gastric ulcers in a subject in need thereof, the methodcomprising administering to the subject therapeutically effective amountof a pharmaceutical composition according to claim
 9. 11. Apharmaceutical composition comprising a derivative according to claim 8or a pharmaceutically acceptable salt, amide, alkyl, or ester thereof,and a pharmaceutically acceptable excipient.